respiratory illness and infections with · the most common illness in infancy and childhood is...
TRANSCRIPT
RESPIRATORY ILLNESS AND INFECTIONS WITH
HUMAN METAPNEUMOVIRUS AND RESPIRATORY
SYNCYTIAL VIRUS IN YOUNG CHILDREN
Marie-Louise von Linstow, MD
Department of Paediatrics
Hvidovre Hospital
PhD thesis
Faculty of Health Sciences
University of Copenhagen
2008
2
RESPIRATORY ILLNESS AND INFECTIONS WITH
HUMAN METAPNEUMOVIRUS AND RESPIRATORY
SYNCYTIAL VIRUS IN YOUNG CHILDREN
Marie-Louise von Linstow, MD
Department of Paediatrics
Hvidovre Hospital
PhD thesis
Faculty of Health Sciences
University of Copenhagen
2008
3
The thesis was handed in on 17 October 2007 for evaluation at the University of Copenhagen, and
accepted on 17 December 2007 for public defence of the PhD degree. The evaluators were:
Henrik Bindesbøl Mortensen, professor, DMSc,
Department of Paediatrics, Glostrup Hospital, Glostrup, Denmark
Peter Oluf Schiøtz, professor, DMSc,
Department of Paediatrics, Aarhus University Hospital, Skejby Sygehus, Aarhus, Denmark
Tine Brink Henriksen, associate professor, PhD,
Department of Paediatrics, Aarhus University Hospital, Skejby Sygehus, Aarhus, Denmark
1
The present thesis is based on the following papers:
I. von Linstow ML, Larsen HH, Eugen-Olsen J, Koch A, Meyer AM, Westh H, Lundgren
B, Melbye M, Høgh B. Human metapneumovirus and respiratory syncytial virus in
hospitalized Danish children with acute respiratory tract infection. Scand J Infect Dis
2004:36;578-84.
II. von Linstow ML, Eugen-Olsen J, Koch A, Winther TN, Westh H, Høgh B. Excretion
patterns of human metapneumovirus and respiratory syncytial virus among young
children. Eur J Med Res 2006;11:329-35.
III. von Linstow ML, Holst KK, Larsen K, Koch A, Andersen PK, Høgh B. Acute
respiratory symptoms and general illness during the first year of life: A population-based
birth cohort study. Submitted.
IV. von Linstow ML, Høgh M, Nordbø SA, Eugen-Olsen J, Koch A, Høgh B. A community
study of clinical traits and risk factors for human metapneumovirus and respiratory
syncytial virus infection during the first year of life. Eur J Pediatr (submitted).
Supervisors
Birthe Høgh, professor, MD, DMSc,
Department of Paediatrics 531, Hvidovre Hospital
Anders Koch, MD, PhD,
Department of Epidemiology Research, Statens Serum Institut
Jesper Eugen-Olsen, MSc, PhD,
Clinical Research Centre, Hvidovre Hospital
2
Acknowledgements
The present thesis is based on studies carried out during my employment as a research fellow at the
Department of Paediatrics, Copenhagen University Hospital, Hvidovre from 2003 to 2007.
I would like to express my gratitude to the following persons who contributed to the studies and the
thesis in various ways:
First of all I would like to thank my supervisors, Birthe Høgh, Anders Koch, and Jesper Eugen-
Olsen for their great help and inputs through the various phases of the study. In particular, I would
like to thank Birthe for her ever-optimistic engagement in my projects, for having confidence in me,
for always being there when needed, and for her admirable ability to focus on the positive sides of
any situation.
Second most, I wish to thank Karina Larsen, who was employed as a study nurse throughout the
clinical phases of the studies and among many other tasks did a wonderful job performing more
than 1300 home visits on a bicycle year-round, always with a smile and an energy that most people
would envy.
I would also like to thank Gorm Lisby, Mette Høgh, and Bente Schirakow from the Department of
Clinical Microbiology, Hvidovre Hospital for setting up analyses for the project, and Anja
Stausgaard, Bodil Landt, Ina Steen Andersen, Jonas Michelsen, Anne-Marie Meyer, and Rikke
Lyngaa Skou for helping perform the laboratory analyses and for handling all the specimens.
Thank you to the staff at the Department of Paediatrics, Hvidovre Hospital, for their positive
attitude to the projects even in busy times. I have only met helpful and kind people during my years
at the following departments at Hvidovre Hospital: the post-natal ward, Clinical Research Centre,
Department of Clinical Microbiology, Department of Clinical Biochemistry, and The Sterile and
Materials Processing Department.
I also thank Klaus Larsen and Jan Wohlfahrt for useful discussions on the statistical analyses,
Nanna Lietmann for helping perform a number of home visits in the most busy period of the study,
3
Niels Steen Krogh for design of the large database, and Yoshio Suzuki for computerizing large
amounts of data into the database.
Furthermore thanks are due to my co-authors Hans Henrik Larsen, Henrik Westh, Bettina
Lundgren, Mads Melbye, Thilde Nordmann Winther, Klaus Kähler Holst, Per Kragh Andersen, and
Svein Arne Nordbø for their valuable contributions to the papers of the thesis, and to Annemette
Kristensen for proof-reading two of the manuscripts and the thesis.
Warm thanks to Lene Nielsen, Tina Sørensen, Anne Brødsgaard, Christian Jakobsen, and Pernille
Fog Svendsen for good company on an otherwise lonely ride.
A very special thanks goes to all the children and their parents who contributed to this thesis by
volunteering to participate in the studies.
The studies were supported by grants from H:S Central Research Fund, Hvidovre Hospital’s
Research Fund, the Pharmacists’ Foundation, the Danish Lung Association, Dagmar Marshall’s
Foundation, the Augustinus Foundation, the Research Fund of Queen Louise’s Children’s Hospital,
Rosalie Petersen’s Foundation, Ebba Celinder’s Foundation, the Gangsted Foundation, and Aase
and Ejnar Danielsen’s Foundation for which I am very grateful. I also wish to thank Johan Lantto
from Symphogen A/S who kindly provided material for the RSV ELISA analyses, Bert Niesters
from Erasmus Medical Centre, Rotterdam, The Netherlands, for providing material for the RSV
PCR analyses, and Phadia ApS, Denmark, for performing the Phadiatop Infant analyses.
Finally, I wish to thank my beloved family: Jørgen, Johanna and Christoffer for every day
reminding me of the most important things in life. I’m sure that one day Johanna will forgive me for
letting her nose be the first to test the nasal swabs.
4
Abbreviations
ARTI Acute respiratory tract infection
RSV Respiratory syncytial virus
LRTI Lower respiratory tract infection
hMPV Human metapneumovirus
APV Avian pneumovirus
URTI Upper respiratory tract infection
n-CPAP Nasal-continuous positive airway pressure
(RT)-PCR (Reverse transcription)-polymerase chain reaction
IFA Immunofluorescence assay
EIA Enzyme immonoassays
GA Gestational age
NPA Nasopharyngeal aspirate
PBS Phosphate-buffered saline
OD Optical density
RT Room temperature
Ct Threshold cycle
nd Not done
na Not available
S.T.A.R Stool transportation and recovery
FRET Fluorescence resonance energy transfer
PDV Phocine Distemper Virus
ELISA Enzyme-linked immunosorbent assay
M-PBS PBS with 2% skim-milk powder
HRP Horseradish peroxidase
TMB Tetramethyl benzidine
SD Standard deviation
PAU Pharmacia Arbitrary Units
OR Odds ratio
RR Relative risk
5
IQR Inter quartile ranges
CI Confidence interval
GEE Generalised estimating equations
SPT Skin prick test
6
Contents
1. INTRODUCTION…………..…………………….…………………………………………….9
2. BACKGROUND………………………………………………………………………………...9
2.1. ILLNESS IN INFANCY………………...………………………….……………………………..9
2.2. RESPIRATORY VIRUSES………………………………………………………………………10
2.3. PARAMYXOVIRIDAE FAMILY…………………………………………………………………..10
2.4. RESPIRATORY SYNCYTIAL VIRUS…………………………………………………………….11
2.4.1. Epidemiology and clinical manifestations……………………………………………11
2.4.2. Transmission………………………………………………………………………….12
2.4.3. Pathogenesis and immunity…………………………………………………………..12
2.4.4. Risk of severe disease…………………………………………………………………13
2.4.5. RSV and atopic disease……………………………………………………………….13
2.4.6. Prevention and treatment…………………………………………………….………13
2.5. HUMAN METAPNEUMOVIRUS………………………………………………………………...14
2.5.1. Discovery of an unknown virus………………..…………………………………...…14
2.5.2. Epidemiology and clinical manifestations…………………………………………....14
2.5.3. Pathogenesis, transmission and immunity………………………………………...….15
2.6. LABORATORY DIAGNOSIS OF RSV AND hMPV…………………………………………..….15
3. AIMS………………………………………………………………………………..………..…16
4. METHODS…………………………………………………………………………………..…17
4.1. THE STUDY AREA…………………………………………………………………………....17
4.2. STUDY POPULATIONS………………………………………………………………………..17
4.3. CLINICAL DATA………………………………………………………………………...…...19
4.4. DEFINITIONS OF ILLNESS EPISODES…………………………………………………………..19
4.4.1. Definitions used in Paper I…………………………………………………………...19
4.4.2. Definitions used in Paper III………………………………………………………….20
4.4.3. Definition used in Paper IV……….………………………………………………….20
4.5. SPECIMEN COLLECTION……………………………………………………………………...20
4.5.1. NPA………….………………………………………………….…………………….20
4.5.1.1. Protein counts in NPAs…………………………………………………………21
4.5.2. Nasal swabs…………………………………………………….……………………..21
7
4.5.2.1. Validation of nasal swabs……………………………………………………….21
4.5.3. Saliva…………………………………………………………………………………23
4.5.4. Blood………………………………………………………………………………….23
4.5.5. Sweat……………………………………………………………….…………………23
4.5.6. Urine………………………………………………………………………………….23
4.5.7. Faeces…………………………………………………………….…………………..24
4.6. REAL-TIME RT-PCR………………………………………………………………….……..24
4.7. ELISA………………………………………………………………………………………25
4.7.1. RSV ELISA……………………………….…………………………………………...25
4.7.2. HMPV ELISA.................................................................…........................................…26
4.8. PHADIATOP INFANT ANALYSIS………………………………………………………………26
4.9. SAMPLE SIZE CALCULATION (BIRTH COHORT STUDY)………….……..………….….……….26
4.10. STATISTICAL METHODS……………………………………………………………………...27
4.11. ETHICAL CONSIDERATIONS………………………………………………………………….28
5. RESULTS………………………………………………………………………………………29
5.1. PAPER I………………………………………………………..…………………………….29
5.2. PAPER II………………………………………………………………………………..……30
5.3. PAPER III…………………………………………………………………………………….32
5.4. PAPER IV…………………………………………………………………………………….34
5.5. PHADIATOP INFANT………………………………………………………………………….35
6. DISCUSSION……………………………………………..……………………………………36
6.1. STUDY DESIGNS, DATA QUALITY AND VALIDITY…………………………………………….36
6.2. SPECIMEN COLLECTION……………………………………………………………………...37
6.3. LABORATORY METHODS USED IN THE STUDIES………………………………….………….. 38
6.4. DISCUSSION OF FINDINGS IN EACH STUDY…………………………………………………...39
6.4.1. Paper I………………………………………………………………………………..39
6.4.2. Paper II……………………………………………………………………………….40
6.4.3. Paper III………………………………………………………………………………41
6.4.4. Paper IV………………………………………………………………………………42
6.4.5. IgE-sensitisation………………………………………………………………………43
7. CONCLUSION AND PERSPECTIVES…………………….……………………………….44
7.1. CONCLUSION………………………………………………………………………………...44
8
7.2. PERSPECTIVES……………………………………………………………………………….44
8. SUMMARY…………………………………………………………………………………….45
9. RESUMÉ PÅ DANSK…………………………………………………………………………47
10. REFERENCES………………………………………………………………………………...49
11. APPENDIX. HEALTH DIARY………………………………………………………………61
PAPER I……………………………………………………………………………………………62
PAPER II………………….………………………………………………………………………..70
PAPER III………………………………………………………………………………………….78
PAPER IV………………………………………………………………………………………...104
9
1. Introduction
Acute respiratory tract infection (ARTI) in infants and young children is a significant public health
problem worldwide. A substantial part of respiratory tract infections is associated with viruses, and
although rarely fatal in industrialised countries, they are a source of significant morbidity and carry
a considerable economic burden.
Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection (LRTI) in
young children17
. Only about 40% of children suspected to have an RSV infection have a positive
test result and in many cases – up to 50% - the etiological agent remains unknown18
, which is in
part due to diagnostic limitations. A proportion of ARTIs may be caused by still unknown
pathogens. In 2001, a hitherto unknown virus was isolated from respiratory specimens from
children with ARTI in the Netherlands, and the virus was named human metapneumovirus
(hMPV)136
. The discovery of hMPV forms the basis of the studies in the present thesis. As the
epidemiology and clinical impact of RSV infection are well described, infections due to hMPV and
RSV were compared in the studies.
2. Background
3.1. Illness in infancy
The most common illness in infancy and childhood is ARTI. The epidemiology of ARTI in infants
differs from that of older children and adults. Early childhood is a time of particular susceptibility to
respiratory ailments, and infants often develop more severe symptoms when infected with a
respiratory virus. This is mainly due to immune immaturity and the smaller anatomy of the lower
airways in infants. Most studies report incidences of 4-7 respiratory episodes per year, depending on
the design of the study, demographics of the populations studied, definitions of respiratory illnesses,
and the methods of surveillance employed25, 74, 81, 85, 96
. Several risk factors for severe LRTI in
developed countries have been described, such as daycare attendance67, 68, 78, 132
and lack of
10
breastfeeding19
. In Denmark, this has led to recommendations of a longer maternity leave and an
aggressive breastfeeding policy. Other risk factors such as crowding and siblings, passive smoking,
low socioeconomic status, psychosocial factors, male gender, and low birth weight are in many
studies also found to be associated with lower respiratory tract disease, while other studies do not
report such associations43
.
Risk factors for upper respiratory tract infections (URTI), which are even more prevalent among
children and have substantial impact on the disease burden experienced by families, have only been
sparsely investigated68, 85
. Likewise, not much is known about minor general illness in infancy as
parents deal with most of these symptoms without seeking medical advice59
.
3.2. Respiratory viruses
A large number of viral pathogens belonging to different virus families have been associated with
ARTI in humans; RSV, hMPV, parainfluenza virus, influenza virus, adenovirus, coronavirus OC43
and 229E, rhinovirus and enterovirus. Since the discovery of hMPV several new viruses have been
identified as a cause of ARTI in humans; SARS-CoV, coronaviruses HKU1 and NL63, human
bocavirus and several parechoviruses. In the following sections only RSV and hMPV, both
members of the family Paramyxoviridae, will be described in detail.
3.3. Paramyxoviridae family
Paramyxoviruses are enveloped, cytoplasmic viruses containing non-segmented, single-stranded,
negative-sense RNA. The Paramyxoviridae family consists of several major pathogens and is
divided into two subfamilies, the Paramyxovirinae and the Pneumovirinae (Figure 1). The
Pneumovirinae subfamily has two genera: the pneumoviruses and the metapneumoviruses. The
classification of the two genera is based primarily on their gene constellation: metapneumoviruses
lack two non-structural proteins, and the gene order is different from that of pneumoviruses. Human
RSV is the type species of the genus Pneumovirus and was until the discovery of hMPV the only
human pathogen identified in the subfamily Pneumovirinae. Avian pneumovirus (APV) causes
URTI in turkeys and other birds and was the sole member of the Metapneumovirus genus until
2001.
11
Figure 1. Classification of viral pathogens of the Paramyxoviridae family that infect humans and
avian pneumovirus (APV) which causes upper respiratory tract illness in turkeys. APV is the
pathogen most closely related to human metapneumovirus (hMPV). Respiratory syncytial virus is
the human pathogen closest related to hMPV.
3.4. Respiratory syncytial virus
3.4.1. Epidemiology and clinical manifestations
Respiratory syncytial virus was first isolated in 1956 from a chimpanzee, and its infectivity in
humans was documented the next year14
. RSV has a worldwide distribution and is one of the main
causes of ARTI. In temperate climates, RSV produces yearly outbreaks from winter to early
spring94
. Two subgroups of RSV exist and both strains circulate concurrently52
. Approximately 2/3
of all children acquire RSV infection during their first year of life; virtually all children have been
infected by 24 months of age, and about half have experienced two infections42
. Primary RSV
infection mostly causes a febrile URTI and is commonly associated with acute otitis media10, 106
, but
up to one third of primary infections also involve the lower airways42
. In young infants, the clinical
manifestations may be non-specific, presenting with poor feeding, irritability, lethargy, or
apnoea128
. In addition to respiratory symptoms, severe RSV infection has been associated with
extrapulmonary symptoms, and RSV or its genetic material have been isolated from cerebrospinal
fluid, myocardium, liver, and peripheral blood indicating a systemic spread at least in some
individuals30
.
Paramyxoviridae
Paramyxovirinae Pneumovirinae
Rubulavirus Respirovirus Morbillivirus Pneumovirus Metapneumovirus
Parainfluenza
types 2 and 4
Mumps Measles Parainfluenza
types 1 and 3
Respiratory
syncytial virus
Human meta-
pneumovirus
Species
Avian
pneumovirus
Genus
Family
Subfamily
12
A study from east Denmark found that 3.4% of RSV-infected children below the age of 6 months
required hospitalisation during the winter season 1995-199672
. A recent study from the US reports
the annual hospitalisation rate to be around 2.5%80
. In affluent countries, very few previously
healthy children suffer life-threatening infections, and deaths are practically confined to those who
are immunocompromised or who have preexisting cardiorespiratory disease94
. However, due to the
common occurrence of RSV infection, even a low mortality rate may have a marked impact on the
total mortality of young children.
3.4.2. Transmission
RSV is highly transmissible: exposure to an RSV-infected infant over 2-4 hours results in infection
among 71% of the exposed individuals46
, and in a family study of RSV-infected infants, 46% of the
exposed family members became infected49
. Close contact appears to be necessary for infection to
spread from one person to another; in one study, no one sitting at a distance greater than 1.8 m from
RSV-infected infants became infected46
. Spread occurs through large droplets of respiratory
secretions or through direct contact with contaminated surfaces where live virus can survive for up
to six hours48
. RSV is secreted in nasopharyngeal secretions usually for 4-7 days45, 49
. However,
some infants may shed virus for up to four weeks and immunocompromised individuals for many
months, thus increasing its contagious nature47
. Older children and adults usually shed virus for 2-4
days51
.
3.4.3. Pathogenesis and Immunity
The incubation period of RSV-induced respiratory disease is 3-5 days126
. Inoculation of upper
respiratory tract epithelial cells occurs primarily through the eyes or nose with subsequent cell-to-
cell transfer of the virus to the lower respiratory tract where it may produce bronchiolitis and/or
pneumonia21
.
Immunity to RSV following natural infection is transient and imperfect, and re-infections are seen
throughout life51, 57
. Both serum and secretory antibodies play a role in the protection against RSV
infection. Resistance to RSV infection in the upper respiratory tract is most likely mediated by local
secretory IgA, whereas serum IgG neutralising antibodies provide protection in the lower
respiratory tract126
. Young infants mount poor antibody responses to primary infection, which may
be secondary to the relative immunologic immaturity of the infants and/or to a suppressive effect of
13
maternally transmitted transplacental antibodies98
. Although complete protection against infection
may not exist, RSV-specific immunity can protect against severe LRTI. Furthermore, cellular
immune responses are thought to play an important role in clearing RSV.
3.4.4. Risk of severe disease
High-risk groups for severe RSV disease are: children born prematurely15, 99
; children with
concurrent heart or lung disease37, 99
; immunocompromised children50
; age<6 weeks99
; and the
elderly35
. In addition, crowding in the home, siblings, daycare attendance, passive smoking, low
birth weight, month of birth, male sex, and a family history of asthma and atopy have been
associated with severe RSV disease15, 55, 58, 77, 103, 116, 134
. Some, but not all studies, find a protective
effect of maternal antibody level and breastfeeding58, 112
. To our knowledge, no studies have
investigated risk factors for RSV-associated upper respiratory illness.
3.4.5. RSV and atopic disease
Whether RSV infection at a young age is causal in the development of atopic disease like asthma
and allergy remains a subject of debate. In most studies, the prevalence of asthma, wheezing, and
IgE sensitisation after RSV bronchiolitis has been higher in the index than the control group119, 123-
125. Some studies find that this association declines over time
71, 111, 129, and according to a Finnish
study early RSV hospitalisation results in reduction of skin prick test positivity 6-10 years after
hospitalisation64
. The potential role of mild RSV infections in infancy in the development of atopic
disease at a later age is even more unclear. Forster et al. have shown that mild RSV infections at a
young age can promote aeroallergen sensitisation during the first year of life, but they were unable
to show atopic manifestations during the first two years of life38
. Finally, it could also be the other
way around; that atopic disposition, wheezing and atopic disorders are determinants for RSV
hospitalisation131
.
3.4.6. Prevention and treatment
Despite many efforts, at present no safe and effective vaccine against RSV infection is available. A
humanised monoclonal antibody (palivizumab) has been approved for its use in high-risk infants to
protect against serious disease due to RSV, though it does not prevent infection. Thorough
handwashing is the most effective method of interrupting transmission of RSV in the homes, in
daycare institutions, and in hospital environments12, 62, 117, 118
.
14
Treatment of RSV bronchiolitis is supportive with supplemental oxygen therapy or nasal-
continuous positive airway pressure (n-CPAP), suction of secretions from the airways, and
sufficient fluid therapy. Antibiotics, corticosteroids, bronchodilator drugs, ribavirin, and
physiotherapy are not recommended routinously44
.
3.5. Human metapneumovirus
3.5.1. Discovery of an unknown virus
In 2001, Dutch researchers isolated an agent from respiratory specimens collected during a 20-year
period from 28 young children with ARTI136
. The agent induced cytopathic effects on cultured
cells, and electron microscopy revealed viral-like particles, but immunological assays using virus-
specific antibodies and polymerase chain reaction (PCR)-based methods using specific primers for
known viral pathogens failed to identify this agent. Experimentally infected monkeys developed
URTI and the virus could be recovered from infected animals. Ultimately, genome sequences of this
novel pathogen were obtained by using a molecular biology technique known as randomly
arbitrarily primed PCR. On the basis of morphological, biochemical, and genetic features, hMPV
was found to be closely related to APV and was tentatively classified in the Metapneumovirus
genus of the Paramyxoviridae family. Phylogenetic analysis revealed the existence of 2 genotypes
of hMPV. Screening of banked human sera showed that hMPV had been circulating for at least 50
years, suggesting that the virus did not recently "jump" to the human population from an animal
reservoir such as birds136
.
3.5.2. Epidemiology and clinical manifestations
Since its discovery in 2001, hMPV has been identified as a cause of respiratory tract illness in all
age groups worldwide. In most studies, hMPV accounts for 4-8% of the ARTI cases in hospitalised
children6, 9, 31, 32, 39, 93, 97, 138
but frequencies up to 21% have been reported24
. In temperate climate
zones, hMPV circulates primarily during late winter/early spring overlapping or following the peak
of RSV activity2, 40, 105, 113, 151
. The clinical symptoms associated with hMPV infections are best
studied in hospitalised children and are very similar to those induced by RSV6, 138, 143, 148
. Most
frequent are cough, fever, coryza, and wheezing, but also symptoms such as diarrhoea, vomiting,
rash, febrile convulsions, conjunctivitis and otitis media have been associated with hMPV
infection6, 39, 107, 127, 137, 149
. Very severe symptoms have been noted in children infected with both
hMPV and RSV in some studies70, 121
but not in others79, 140, 151
. hMPV can cause an influenza-like
15
illness in adults and has been associated with wheezing and asthma in young children31, 32, 63, 92, 107,
138, 149. hMPV is uncommon in asymptomatic children and adults
33, 136, 149. Children with underlying
illnesses such as a history of prematurity, congenital heart or lung disease, or immunodeficiency
may be predisposed to severe hMPV disease65
. However, risk factors for severe or early hMPV
infections have not been thoroughly investigated. Furthermore, information on hMPV infection in
non-hospitalised children is very sparse.
3.5.3. Pathogenesis, transmission and immunity
Animal experiments have shown that hMPV replicates in the respiratory epithelium only3, 73
, and
the current understanding is that during infection both the virus and the disease are limited to the
respiratory tract. However, findings from different groups indicate that hMPV infection is
associated with encephalitis and may thus cause disseminated infection in some individuals41, 66, 120
.
A recent study suggests that the pathogenesis of hMPV-associated LRTI involves co-infection with
pneumococcus, as vaccination with pneumococcal conjugate vaccine reduces the incidence of
hMPV (as well as other respiratory viruses) LRTI hospitalisations in young children88, 89
. The
incubation period of hMPV is unknown but has been estimated to be 4-6 days27
. hMPV infection in
children appears to have a great impact on their families; in one study 12.5% of the family members
of hMPV-positive children had a disease similar to that of the infected child, which was
significantly more than for RSV-positive children8. The shedding period and the modes of
transmission have not yet been investigated, although they are considered similar to those of RSV.
More than 90% seroprevalence has been found by the age of five28, 83, 136
, but like other respiratory
viruses, hMPV infection does not confer lasting immunity and re-infections are seen throughout
life.
3.6. Laboratory diagnosis of RSV and hMPV
Virus isolation from respiratory specimens by cell culture has long been the gold standard for
diagnosis of viral infection. However, cell culture requires specimens to be transported and stored
under ideal conditions, and it takes several days before a diagnosis can be established. hMPV grows
poorly in cell culture, replicates in few cell lines only, and shows late cytopathic effect, which are
some of the reasons for its late identification.
In recent years, reverse transcriptase-polymerase chain reaction (RT-PCR) has proved to be a highly
sensitive and specific method for the diagnosis of RSV and hMPV infections1, 34
. Especially real-
16
time RT-PCR assays are rapid, specific, quantitative, and highly sensitive and are capable of
subtyping RSV in clinical specimens75, 76, 90, 95
. Other methods used for antigen detection are
immunofluorescence assays (IFA) and enzyme immonoassays (EIA), using virus-specific
antibodies. These methods are rapid, but not as sensitive as RT-PCR1, 13, 29, 108
.
Serological tests detecting virus-specific antibodies (ELISA, IFA, neutralization and
haemagglutination inhibition tests) provide immunologic evidence of infection after the acute phase
of the disease and are only useful for epidemiological studies or retrospective diagnosis.
3. Aims
The overall aim of this thesis was to study the clinical epidemiology of hMPV compared with RSV
in hospitalised and non-hospitalised children in the area of Copenhagen.
The specific aims were
1. To determine the frequencies and clinical features of hMPV and RSV infections in children
hospitalised with acute respiratory tract infection (Paper I).
2. To examine the excretion patterns of hMPV and RSV in children (Paper II).
3. To measure the incidence and prevalence of acute respiratory symptoms and overall
morbidity in unselected infants in the community and to identify risk factors for respiratory
symptoms in the first year of life (Paper III).
4. To study the clinical symptoms and risk factors associated with hMPV and RSV infections
in unselected infants in the community during the first year of life (Paper IV).
17
4. Methods
4.1. The study area
The studies in the present thesis involve children residing in the area of Hvidovre Hospital,
Denmark, which serves an area of Copenhagen with 396,000 persons (35% of the total population
of Greater Copenhagen) and has around 5,500 births a year. The first study also includes children
residing in the area of Amager Hospital, which is smaller and serves approximately 158,000
citizens. Both hospitals are located in a city area near the capital with housing of both flats and free
houses, but no country-area. In this area, approximately 14% of 20-45-year-old inhabitants have a
high academic degree and 12% have a medium-long education. In families with children, 52% live
with one child and in 12% of families there are three or more children20
. In Denmark, 27% of the
adult population smoke133
. The climate is temperate.
4.2. Study populations
The studies forming the basis of the present thesis were carried out in three different groups of
children:
1. Prevalence study: The first study included 383 stored routine nasopharyngeal aspirates
(NPA) obtained from 374 children hospitalised with ARTI at the Departments of
Paediatrics, Hvidovre or Amager Hospital, during the RSV seasons (November-May)
1999-2000 and 2001-2002. The NPAs were analysed for the presence of hMPV and RSV
by RT-PCR and the medical records of all children were systematically reviewed.
2. Excretion study: The second study included children admitted to the Department of
Paediatrics, Hvidovre Hospital, during the winter season 2003-2004 and who were tested
positive for hMPV or RSV in a routine NPA. The children were followed for three weeks
with weekly home visits to examine the excretion of viral RNA in different secretions; at
inclusion and each week, one NPA specimen, one saliva sample, one urine sample and one
stool sample were taken from each child. In addition, sweat and blood samples were
obtained at inclusion.
18
3. Birth cohort study: In this study, children were recruited from the post-natal ward at
Hvidovre Hospital. Due to logistic restraints and in accordance with statistical power
calculations approximately 250 healthy newborns were enrolled into the study during a
12-month period from May 2004 to May 2005. To ensure that children were sampled
equally throughout the year approximately 20 children, of whom half had siblings, were
enrolled each month on predesignated weeks. The inclusion criteria for participation in the
study were: Infants free of obvious health problems, and for practical purposes living
within 11 km of Hvidovre Hospital. Exclusion criteria were: Infants whose parents did not
understand or speak Danish or English; infants whose mothers had a serious psychiatric
disorder; infants with congenital diseases; and if change of address to outside the area of
Hvidovre Hospital was planned within 12 months of enrolment. All children were
followed for 12 months with monthly home visits. At every home visit children had a
nasal swab taken. Figure 2 outlines the study design of the birth cohort study.
Inclusion and follow-up Follow-up
↓ ↓* ↓ ↓* ↓ ↓* ↓ ↓* ↓ ↓* ↓ ↓* ↓ ↓* ↓ ↓* ↓ ↓* ↓ ↓* ↓ ↓* ↓ ↓*
Infants
200
100
May 2004 May 2005 May 2006
Figure 2. Study design of birth cohort study.
↓ = Monthly visits (health diary, nasal swab)
* = Every second month (interview)
Blood sample at 5 days and at 12 months of age
19
4.3. Clinical data
The medical records of children included in studies 1 and 2 were systematically reviewed and
information was registered on demographic characteristics, symptoms during hospitalisation,
physical examination, treatment, and final diagnosis. In the excretion study, parents were
interviewed weekly about their child’s present and previous illnesses and related diseases in
household members. In addition, information was obtained on gestational age (GA), duration of
breastfeeding, daycare attendance, smoking in the household, and atopic dispositions.
In the birth cohort study, parents were provided monthly with a health diary displaying 12 different
symptoms and clinical signs, doctor’s visits, hospital admissions and medicine (see Appendix I). At
the first home visit the parents were interviewed about household members, parents’ education and
employment, ethnicity, birth weight of the included infant, breastfeeding, dispositions (hay fever,
asthma and atopic dermatitis) and exposures (smoking in homes, smoking during pregnancy, pets,
moist, carpets, drying clothes inside). Questions concerning factors that could change over time
were repeated every second month. Ethnicity was defined as Western/non-Western according to the
biologic parents’ place of birth. Only if both parents were born in a Western country, ethnicity was
defined as Western. Socioeconomic status was classified according to the Danish social
classification system choosing the social group, which comes first in the order 1 to 5 in families
where the mother and father were classified in two different categories54
. Allergic dispositions were
regarded as present if the parents answered "yes" to the question "has anyone in the family ever had
asthma, hay fever or atopic dermatitis?" Dispositions by first- and second-degree relatives were
included in the analyses. Children were seen in the hospital for the final interview and blood
sampling during the month they turned one year or the following month.
4.4. Definitions of illness episodes
4.4.1. Definitions used in Paper I:
- ARTI: A period with symptoms and clinical signs of URTI or LRTI.
- URTI: One or more of the following signs present: nasal discharge, cough, a bulging red
tympanic membrane and pharyngo-tonsillar erythema or exudate without signs of LRTI.
- LRTI: Signs of URTI together with chest indrawing and tachypnoea combined with rhonchi
and/or crepitation. All children who required respiratory support were included in the LRTI
group.
20
- Asthmatic bronchitis: Symptoms of ARTI in combination with wheezing and/or rhonchi on
lung auscultation.
- New ARTI episode: An episode of ARTI with a 7-day interval free of symptoms to the
previous ARTI episode.
4.4.2. Definitions used in Paper III:
- ARTI: Nasal discharge together with one or more of the following symptoms: cough, fever,
wheezing, tachypnoea, general malaise, or loss of appetite.
- Simple rhinitis: Nasal discharge as the only symptom.
- New episode: An episode of ARTI or simple rhinitis with a 6-day interval free of symptoms
to the previous same type of episode.
- Time at risk: Days with no recorded symptoms excluding the 6 consecutive days without
symptoms following an episode.
- Incidence: Number of episodes divided by person time at risk.
4.4.3. Definition used in Paper IV:
- Illness episode: Time from first day with symptoms to last day with symptoms without
symptom-free days in the interval. All symptoms occurring during the episode were
considered associated with the hMPV or RSV infection.
4.5. Specimen collection
4.5.1. NPA (Paper II)
NPA specimens were taken using a soft plastic tube sucking secretions from the nasopharynx into a
sterile 10 ml tube. Afterwards, two ml of phosphate-buffered saline (PBS) buffer were suctioned
through the plastic tube, and the secretions were collected in the sterile tube. At home visits, the
same device connected to a foot pump was used (Figure 3).
Figure 3. NPA collection devise connected to a foot pump.
21
4.5.1.1. Protein counts in NPAs (Paper II)
As only a small amount of secretion was obtained from asymptomatic children during the home
visits, the total protein count in the NPA's was determined as a measure of sufficient sample
material was obtained in each case. The protein concentrations in NPAs were determined by using
the BCA Protein Assay Reagent Kit (Pierce, Rockford, IL). Briefly, the assay is based on
Bicinchoninic acid for detection and quantification of four amino acids, cysteine, cystine,
tryptophan, and tyrosine within a sample. The obtained results were quantified relative to a standard
curve based on bovine serum albumin at an optical density (OD) of 562 nm.
4.5.2. Nasal swabs (Paper IV)
Nasal swabs were obtained using a sterile cotton swab on an aluminium shaft, which was dipped
into sterile sodium chloride before applied to the nose. Swabs were collected from a depth of 2 to 3
cm and were then placed into a vial containing 1 ml of viral transport medium (bovine serum
albumin and antibiotics in PBS) (Figure 4 and 5). The aluminium shaft was chosen, as it could be
bended in the preferable angle prior to collection. At some occasions the parents were instructed in
sampling technique and sent the samples by mail to the laboratory.
Figure 4. Cotton swab and viral Figure 5. Collection of a nasal swab.
transport medium.
4.5.2.1. Validation of nasal swabs
The sensitivity of the nasal swabs compared with NPA was evaluated in 20 children aged 2 weeks
to 6 months and hospitalised with RSV infection at the Department of Paediatrics, Hvidovre
Hospital (Table 1).
22
Table 1. Results of real-time RT-PCR analyses of RSV RNA detected in nasopharyngeal aspirates,
nasal swabs collected from a depth of 2-3 cm (nasal swabs), nasal swabs collected superficially
from the nostrils (superficial swab), and nasal swabs stored at room temperature for 24 hours
(swabs RT). All samples were collected from hospitalised children.
Child No. NPA Nasal swabs Superficial swab Swabs RT
result Ct result Ct result Ct result Ct
1 pos* na pos 47.4 pos >51 nd na 2 pos 28.6 pos 34.0 pos 34.9 nd na 3 pos 31.0 pos 43.1 pos 44.9 nd na 4 pos na pos 32.5 pos 47.2 nd na 5 nd na pos 31.0 pos 36.7 nd na 6 pos 19.0 pos 26.9 pos 26.0 nd na 7 pos 24.1 pos 32.5 pos 32.5 nd na 8 pos 20.7 pos 28.6 pos 29.1 nd na 9 pos 21.5 pos 31.1 pos 32.7 nd na 10 nd na pos 28.8 pos 31.8 nd na 11 pos 22.4 pos 26.2 pos 27.8 nd na 12 pos 27.2 pos 27.1 pos na nd na 13 pos 30.6 pos 31.9 nd na pos 32.0
14 pos 26.5 pos 34.4 nd na pos 37.6
15 pos 20.4 pos 22.2 nd na pos 22.5
16 pos 25.1 pos 36.1 nd na neg na
17 pos 23.1 pos 23.4 nd na pos 23.8
18 pos 23.1 pos 28.9 nd na pos 32.2
19 pos 18.5 pos 20.3 nd na pos 22.8
20 pos* na pos 22.3 nd na pos 22.6
* These NPAs were tested by enzyme immunoassay.
NPA = nasopharyngeal aspirate, RT=room temperature, Ct=threshold cycle, nd = not done, na=not available.
Nasal swabs were taken within one day of the NPA. A swab was obtained from each nostril; in 12
children the first swab was taken very superficially and the second as described above. In 8
children, both swabs were collected as described above. One swab from each of these 8 children
remained at room temperature (RT) until the next day, whereas the remaining swabs were stored at
4˚C immediately. In this way, it was tested if the depth of collection and time to cooling had a
major impact on the results of the PCR analyses. Swabs were analysed for the presence of RSV
RNA by real-time RT-PCR as described below. Except for one swab stored at RT for 24 hours,
RSV RNA was present in all samples, although most nasal swabs contained lower amounts of viral
RNA, illustrated by a higher threshold cycle (Ct) value in the swabs.
23
4.5.3. Saliva (Paper II)
Saliva was collected using the Oracol device (Malvern Medical Developments, UK), which is a
cylindrical polystyrene sponge attached to a plastic stick designed to be used as a toothbrush
(Figure 6).
Figure 6. Collection of saliva using the Oracol
device.
4.5.4. Blood (Paper II, IV)
In the excretion study, 1-2 ml blood were collected from hospitalised children who had blood
samples taken due to their illness.
In the birth cohort study, blood sampling from the children was performed as a heal prick together
with routine PKU testing and as venous puncture at age 12 months. Blood samples from the mother
were obtained at enrolment. Samples were taken to obtain exact information about serum RSV and
hMPV antibody levels of the mother and child close to delivery and of the child by the age 12
months.
4.5.5. Sweat (Paper II)
Sweat was collected by pilocarpine iontophoresis (Figure 7). A disc of 0.5 % pilocarpine was
applied to the medial aspect of the forearm after washing thoroughly with sterile water. After
stimulation with iontophoresis for five minutes the arm was again cleaned with sterile water and
sweat was collected through a closed spiral microbore tube (Figure 8).
4.5.6. Urine (Paper II)
Urine was collected using a plastic urine collector bag attached to the outer genitalia and then the
urine was transferred to a sterile 10 ml glass.
24
Figure 7. Pilocarpine iontophoresis. Figure 8. Collection of sweat through
Stimulation of the forearm with iontophoresis. a closed spiral microbore tube, which is
The red disc contains pilocarpine, and applied on the stimulated area for 30
the black disc is a reference. minutes.
4.5.7. Faeces (Paper II)
Faeces were collected from the nappy by a sterile spoon and placed into a stool transportation tube
containing 3-4 ml of Stool Transportation And Recovery (S.T.A.R.) buffer (Figure 9). The S.T.A.R
buffer contains nucleases inactivators and permits storage and shipment of stool specimens for up to
five days at 15-20°C without compromising the stability of RNA.
Figure 9. S.T.A.R buffer and stool collection device.
4.6. Real-time RT-PCR (Paper I, II, IV)
Nucleic acid purifications from NPAs, nasal swabs, saliva, urine and faeces specimens were carried
out on a MagNA Pure LC Instrument. The amount of material used varied according to type of
specimen. RNA from sweat and blood samples was extracted manually. Real-time RT-PCR
25
analyses of hMPV and RSV were performed on a LightCycler instrument (Paper I+II) or on a
TaqMan PCR system (Paper IV).
In Paper I, commercially synthesized primers targeting the hMPV N gene were used for detection of
hMPV RNA87
. However, gradually it became clear that these primers only detected one subtype of
hMPV. In the second paper, we used the same primers but added another primer set, which we were
confident detected both subtypes of hMPV90
. These were used in the fourth paper as well.
In Papers I and II, primers and fluorescence resonance energy transfer (FRET) probes were used for
detection of RSV as described by Whiley et al147
. In paper IV, primer/probe mixes for detection of
RSV A and RSV B were ready-to-use mixtures obtained from Dr. Bert Niesters, Erasmus Medical
Centre, Rotterdam, The Netherlands.
In all real-time RT-PCR analyses, standard precautions were taken throughout the PCR process to
avoid possible cross and carry-over contamination. Positive and negative controls were included in
purification and PCR procedures. For samples run on the TaqMan, Phocine Distemper Virus (PDV)
was added to each sample prior to purification as an internal control. All hMPV and RSV-positive
samples identified in Paper IV were tested for co-infection with rhinoviruses, adenovirus, influenza
A and B, parainfluenzaviruses 1-3, coronaviruses OC43, 229E, NL63, and HKU1, and human
bocavirus by PCR using the TaqMan system.
4.7. Enzyme-linked immunosorbent assay (ELISA) (Paper IV)
4.7.1. RSV ELISA
ELISA was used to determine anti-RSV IgG levels in plasma from mothers at the time of delivery
and from their one-year-old children. Ninety-six-microwell, high-binding ELISA plates were coated
with purified RSV antigen (8RSV79, HyTest, Finland) and incubated overnight at +4C. The plates
were washed with PBS containing Tween-20 and blocked with PBS containing 2% skim milk
powder (M-PBS). Plates were washed, and serial dilutions of the positive control (3.86 mg/ml) were
added together with plasma samples diluted in M-PBS and a negative control. Samples were
measured in duplicate. After washing, plates were incubated with horseradish peroxidase (HRP)-
conjugated goat-anti-human IgG, and antibodies were detected by adding 3,3´, 5,5´- tetramethyl
benzidine (TMB) substrate. The reaction was stopped by using 1 M H2SO4 and the extinction was
measured at 450 nm.
26
4.7.2. hMPV ELISA
hMPV antigen and positive and negative controls were obtained from Dr. Svein Arne Nordbø,
Department of Medical Microbiology, Trondheim University Hospital, Trondheim, Norway. In
Norway, hMPV antigen was prepared by sonicating infected LLC-MK2 cells in carbonate buffer.
Sonicated cell pellet from the same cell line negative for hMPV was prepared the same way and
used as negative control antigen. As positive and negative controls sera from two anti-hMPV IgG-
positive persons and two anti-hMPV IgG-negative persons were used, respectively.
The ELISA plates were coated in every second well with hMPV antigen and negative control
antigen, respectively, and incubated overnight at +4C. The plates were washed with PBS
containing Tween-20. Plasma samples and negative controls, diluted in M-PBS, were added in
addition with serial dilutions of the positive control. After washing, plates were incubated with
HRP-conjugated goat-anti-human IgG, and the presence of hMPV antibodies was detected by
adding TMB substrate and then 1 M H2SO4 to stop the reaction. The extinction was measured at
450 nm with a reference filter at 630 nm. Net OD values were calculated as OD values for the
antigen minus OD values for the control antigen. The cut-off value of each hMPV and RSV assay
was defined by adding 2 standard deviations (SD) to the mean OD of all negative sera.
4.8. Phadiatop Infant analysis
Children in the birth cohort study were tested for the presence of allergen-specific IgE antibodies5.
The analyses were performed by Phadia ApS, Denmark. Phadiatop Infant was analysed
quantitatively in plasma, collected at one year of age, using Pharmacia CAP SystemTM
. The
following allergens were included in the test; Egg white, cow's milk, peanut, Dermatophagoides
pteronysssinus, cat, dog, birch, timothy, and mugwort. Values ≥ 0.35 Pharmacia Arbitrary Units per
litre (PAU/l) were considered to indicate sensitisation and were coded as positive.
4.9. Sample size calculation (birth cohort study)
We assumed that 75% and 25% of the children became infected with RSV and hMPV, respectively,
during the first year of life and that the cases were detected during the study period. With a chosen
sample size of 200 subjects it would be possible to detect differences in symptoms related to RSV
and hMPV infection with an odds ratio (OR) of 2.8 under the assumption of 5% significance level
and 80% strength. Risk factors for RSV or hMPV infection during the first year of life could be
detected with relative risks (RR) of 1.3 and 2.1, respectively, under the assumption that the actual
27
risk factors were time-independent and present in 50% of the population. Power calculations were
made in EpiInfo 2000.
4.10. Statistical methods (Paper I, II, III, IV)
Differences in categorical data between the various groups of children were tested by using chi-
square test or Fisher's exact test. For non-normally distributed continuous data, median, range, and
in some cases inter quartile ranges (IQR), were calculated and compared by using the non-
parametric Mann-Whitney U-test and the Kruskal-Wallis test. Shedding durations of RSV and
hMPV RNA in NPAs were estimated by Kaplan-Meyer curves and differences were evaluated by a
log rank test.
In Papers III and IV, logistic regression analyses were performed to assess the importance of
various factors for dichotomic outcomes, and to calculate OR. To reduce problems with co-
linearity, only 1-3 variables from each group of covariates were included. Effect modification was
evaluated in logistic regression analyses with interaction terms.
In Paper III, logistic regression was used to model the daily episode status. For incidence only days
at risk were modeled and for prevalence all observed days were included. Relevant information
from interview schemes was carried forward two months. To account for the possible correlation
between episodes from the same child, OR and confidence intervals (CI) were estimated by using
generalised estimating equations (GEE) with an autoregressive correlation structure (AR(1)). P-
values were calculated by using Wald’s test.
All regression analyses in Paper III were adjusted for sex, age and calendar period. The calendar
effect was modeled as a periodic function with a period of one year by inclusion of a sine and a
cosine term in the models. The parameters for these two terms were transformed into parameters
giving the time of maximum incidence/prevalence and the OR for December vs. July, respectively,
and standard errors of these estimates were calculated.
Multiple logistic regression analyses were performed by including all the selected predictor
variables in a model and successively removing the variable with the highest p-value, continuing
until all predictors had a p-value below 0.1. A p-value<0.05 was considered significant. Depending
28
on the project, data were analysed by using the SAS software version 8e, SPSS version 12.0 or 13.0
for Windows, or R version 2.4.1 by using the geepack library.
4.11. Ethical considerations
The studies fulfilled the Helsinki Declaration II and were approved by The Ethics Committee of
Frederiksberg, Copenhagen, Denmark (J. No. 01-028/03, 01-190/03, and 01-049/04), and by the
Data Protection Board in Denmark (J. No. 2003-41-3092, 2003-41-3469, and 2004-41-4147).
29
3 11 21 34 58 45 30 1 *
5. Results
5.1. Paper I
In the first study, hMPV was identified in NPAs from Danish children hospitalised with ARTI,
thereby proving that hMPV circulates in Denmark and may be a cause of respiratory illness. hMPV
was detected in 11 (2.9%) and RSV in 190 (49.6%) of 383 samples. Two children were co-infected
with both hMPV and RSV. These two children were not more severely ill than mono-infected
children and were left out of statistical analyses. Age at admission was median 3.5 and 3.2 months
for hMPV and RSV-positive children respectively. The peak incidence of hMPV infection was in
February, approximately one month later than the RSV peak (Figure 10).
Figure 10. Seasonal distribution of hMPV and RSV-positive ARTI episodes during 2 winter
seasons. *Total number of NPA examined per month.
1 12 16 35 71 41 2 0 *
2001-2002
0
20
40
60
80
100
Oct Nov Dec Jan Feb Mar Apr May
% in
fecte
d
hMPV
RSV
1999-2000
0
20
40
60
80
100
Oct Nov Dec Jan Feb Mar Apr May
% in
fecte
d
hMPV
RSV
30
Overall symptoms and clinical findings were similar among hMPV and RSV-positive episodes.
However, no hMPV-infected children required treatment with n-CPAP or supplemental tube
feeding in contrast to a quarter of the RSV-positive children (Table 2). The distribution of clinical
diagnosis (LRTI or URTI) in hMPV and RSV-infected children was similar. However, when
looking specifically at those of the children in the LRTI group with a diagnosis of asthmatic
bronchitis, 6 (66.7%) of the hMPV-positive children received this diagnosis compared with 20
(10.6%) of the RSV-positive children (p<0.001) (Table 2).
Table 2. Symptoms, clinical findings, treatment, and main diagnoses in children hospitalised with
respiratory tract infection, Copenhagen, 1999/2000 and 2001/2002
hMPV (n=9) RSV (n=188) p-value
No. positive/
total
(percent) No. positive/
total
(percent) hMPV vs. RSV
Symptoms and clinical signs
Reported fever 6/8 (75.0) 127/184 (69.0) 1
Nasal discharge 6/6 (100) 145/151 (96.0) 1
Cough 8/8 (100) 175/175 (100) -
Vomitus or diarrhoea
0/8 (0) 38/186 (20.4) 0.36
Tachypnoea 8/9 (88.9) 146/180 (81.1) 1
Chest indrawing 7/8 (75.0) 109/180 (60.6) 0.49
Rhonchi 7/9 (77.8) 88/187 (47.1) 0.09
Crepitation 1/9 (11.1) 77/187 (41.2) 0.09
Treatment
Nasal CPAP 0/9 (0) 50/188 (26.6) 0.12
Nasal O2 1/9 (11.1) 49/188 (26.1) 0.45
Assisted ventilation 0/9 (0) 2/188 (1.1) 1
Tube feeding 0/9 (0) 55/188 (29.3) 0.06
Main episode diagnosis
Upper respiratory tract infection 1/9 (11.1) 38/188 (20.2) 0.7
Lower respiratory tract infection 8/9 (88.9) 145/188 (77.1) 0.7
Asthmatic bronchitis
6/9 (66.7) 20/188 (10.6) <0.001
5.2. Paper II
In this study, 44 children aged 0.5-38 months were included (37 RSV-positive, 6 hMPV-positive,
and 1 co-infected child). The hMPV and RSV-infected children did not differ significantly
according to ethnicity, gender, duration of symptoms prior to admission, hospitalisation time, GA,
chronic diseases, number of siblings, and smoking in household. Median age was 3.7 months for
RSV-positive children and 7.8 months for hMPV-positive children (p=0.1).
Viral RNA was detected in saliva from 26/38 (68%) RSV-infected children and in 1/7 (14%)
hMPV-infected children. Eighty-nine percent of the positive saliva samples were taken within the
31
first six days after diagnosis. RSV RNA was found in five stool samples from five different
children. All positive samples were taken within two days of the diagnostic NPA, and four of the
children had diarrhoea. hMPV RNA was not detected in stools. Three RSV-positive children (9%)
and two hMPV-positive children (29%) shed viral RNA in sweat including the child with co-
infection, who shed both RSV and hMPV in sweat. Children shedding RNA in sweat were either
less than five weeks of age or had a chronic lung disease. We did not detect viral RNA in urine or
blood samples.
Nine of 12 children shedding RSV RNA after 2 or 3 weeks had a negative RSV PCR test result in
between. Melting point temperature analyses of NPAs from six of these children showed that they
were infected with the same subtype of RSV (A or B) in all their positive samples. Assuming that
all children shed RSV until the last positive sample, the median duration of RSV shedding was 11.5
days (Inter-quartile range (IQR) 6.5-18.5) while the median duration of hMPV shedding was 5.0
days (IQR 3.5-10.0) (p=0.001, Figure 11). There was a statistically significant association between
protein count in the NPAs and symptoms at the time of sample collection (p<0.0001), between
protein count and a positive test result (p=0.01), and between protein count and time of NPA
collection (p<0.01). Anyhow, this could not explain all the negative test results in between two
positive test results, as only three of nine children had the lowest protein count in their negative
sample.
Figure 11. Kaplan Meyer analysis of
shedding duration of RSV and hMPV
RNA in nasopharyngeal aspirate
specimens. RSV: median 11.5 days
(IQR 6.5-18.5), hMPV: median 5.0
days (IQR 3.5-10.0), p=0.001
32
Fever
Wheezing
Fast breathing
Conjunctivitis
Hoarseness
Skin rash
Vomiting
Diarrhoea
Nasal
discharge
Lost appetite
Cough
General
malaise
More than 75% of family members of both RSV and hMPV-infected children had a URTI when
they were followed up. In families with more than one child, an older sibling was the first person to
present symptoms in more than two thirds of the cases (75% for RSV and 60% for hMPV).
5.3. Paper III
Of 242 children whose parents accepted to participate in this study, 228 children were included in
the analysis with an observation period of 80,013 days. Of these 228 children, 121 were boys and
105 had siblings. Twenty-three percent of the children lived with parents who smoked and 72%
belonged to social class 1 or 2. Breastfeeding was initiated by 95% of the mothers. Only 22% of the
infants attended daycare outside the home by the age of 10 months.
On average, children had one or more symptoms for 3.5 months during their first year of life, nasal
discharge and cough being far most prevalent (Figure 12). Frequency of all symptoms increased
steeply after six months of age. Each child had on average 6.3 episodes (median=5.1, IQR: 3.3-7.8)
of ARTI and 5.6 episodes (median=4.3, IQR: 2.1-7.3) of simple rhinitis per 365 days at risk. Forty
percent of simple rhinitis episodes proceeded into an ARTI episode. An average ARTI episode
lasted for 4.7 days (median=3, IQR: 2-6).
Figure 12. Percentage distribution of general symptoms.
33
The odds of developing ARTI and simple rhinitis increased significantly from six months of age, in
the winter season, and when having siblings aged 1-3 years. Odds of an ARTI were increased for
children attending large daycare centres, while large household size and living in small average
rooms were associated with simple rhinitis (Table 3). The effects of age and season are illustrated in
Figures 13 and 14.
Table 3. Multiple logistic regression analysis for incidence of acute respiratory tract infection
(ARTI) and simple rhinitis. The effects of age and season are illustrated in Figure 13 and 14.
Variable ARTI Simple rhinitis
OR 95% CI p value OR 95% CI p value
Siblings in day nursery (½-3 y)
No
Yes
1.00
1.41
Reference
(1.08-1.84)
0.01
1.00
1.99
Reference
(1.50-2.64)
<0.001
Each additional person in household - - - 1.23 (1.10-1.38) <0.001
Size of residence
Total m2/no. of rooms > 30 m
2
Total m2/no. of rooms < 30 m
2
-
-
-
1.00
1.26
Reference
(1.02-1.56)
0.03
Daycare attendance
No
Family daycare home (2-4
children)
Daycare centre (10-15 children)
1.00
1.46
1.49
Reference
(0.81-2.61)
(1.10-2.02)
0.02
-
-
-
Variables included in analyses are: age, gender, season, gestational age, mother’s age, ethnicity, mother had a cold<2
weeks prior to delivery, siblings in daycare centre and day nursery, daycare attendance, socioeconomic status, and
carpets. For ARTI included are in addition smoking inside, maternal smoking, breastfeeding, number of adults in the
household, siblings, and atopy in siblings. For simple rhinitis included are in addition smoking during pregnancy,
paternal education, exclusive breastfeeding, atopy in the family, moisture in home, size of residence, household size,
and smoking in household. OR, odds ratio; CI, confidence interval
Figure 13. Multiple logistic regression analyses of age distribution of respiratory symptoms.
34
A: Incidence of simple rhinitis, B: Incidence of acute respiratory tract infection.
Figure 14. Multiple logistic regression analyses of seasonal distribution of respiratory symptoms.
A: Incidence of simple rhinitis, B: Prevalence of simple rhinitis, C: Incidence of acute respiratory
tract infection, D: Prevalence of acute respiratory tract infection.
5.4. Paper IV
Of 242 children whose parents accepted to participate in the study, 217 children were followed
throughout one year and were included in the analysis.
Anti-hMPV IgG was found in 38 (17.5%) children and anti-RSV IgG in 172 (79%) children by age
one year. Odds of being anti-hMPV IgG-positive were increased by a factor 2.36 (95% CI: 1.06-
5.27) and 3.82 (95% CI: 1.75-8.34), respectively, in children born during the spring and in children
with older siblings. Drying clothes inside was associated with a decreased risk of being anti-hMPV
IgG-positive (OR=0.45, 95% CI: 0.21-0.97). Factors associated with being anti-RSV IgG-positive
were: GA<38 weeks (OR=3.39; 95% CI: 1.42-8.05), increasing paternal age (OR=1.85 per five yrs;
35
95% CI: 1.28-2.68), and wall-to-wall carpeting (OR=3.15; 95% CI: 1.29-7.68), while birth into the
spring decreased the odds of being anti-RSV IgG-positive (OR=0.27, 95% CI: 0.09-0.85). Sleeping
outside during the day seemed to protect against RSV infection, however non-significantly
(OR=0.44, 95% CI: 0.19-1.03). Risk factors for RSV hospitalisation (n=11) were: older siblings
(OR=4.49; 95% CI: 1.08-18.73) and smoking in the household (OR=5.06; 95% CI: 1.36-18.76).
Exclusive breastfeeding for the first 14 days of life was associated with a reduced risk of being
hospitalised (OR=0.21, 95% CI: 0.06-0.79).
The time of primary infection was identified in 10 hMPV and 15 RSV infections. RSV-positive
samples were identified from children down to 26 days of age, while primary hMPV infection was
only seen in children older than six months of age. All possible symptoms from the health diaries
occurred in both RSV and hMPV-infected children, although more frequently among the RSV-
positive children (non-significant).
5.5 Phadiatop Infant
Phadiatop Infant was positive in 15 (7%) of the children at age 12 months. Nine children were IgE-
sensitised to milk, seven to egg and one to peanut. Of these children, 40% had atopic dermatitis
compared with 18% of all children (p=0.03). IgE-sensitisation was not associated with hMPV or
RSV infection, respiratory symptoms, diarrhoea or atopic disposition.
36
6. Discussion
6.1. Study designs, data quality and validity
In the first study, we included all children who had been hospitalised with an ARTI during two
winter seasons and for whom a routine NPA for RSV analysis had been stored. According to the
result of the PCR analysis of the NPA, children were divided into three groups (hMPV-positive,
RSV-positive, and hMPV/RSV-negative). Because children in each group were hospitalised with
roughly the same symptoms, in the same hospitals, at the same period of time, the risk of selection
bias was avoided.
In the excretion study, children were included when the result of the NPA test was available and
followed three weeks from then. The results of shedding duration are therefore related to time of
hospitalisation rather than to time of infection. Parents were interviewed weekly on symptoms in
the child and the family minimizing the risk of recall bias. However, in a few cases, the parents
reported the first date with symptoms in their child to be later than the date of hospitalisation, which
may be interpreted as a recall bias. The main purpose of this study was to investigate the sites of
viral excretion. However, when studying the prevalence of respiratory symptoms in family
members it would have been appropriate to have a control group of families with RSV and hMPV-
negative children to see if the same amount of respiratory illness occurred in such families.
The birth cohort study was designed to reduce the effects of chance and bias. The study was
conducted over two years to reduce the impact of possible seasonal variation. Information on
background variables was collected at the first home visit (after one month), and we used diary
cards to be filled out on a daily basis, thereby minimizing recall bias11, 142
. The active surveillance
by monthly home visits ensured that any queries concerning the diaries were rectified, it helped
maintain high compliance by parents and resulted in a low drop-out rate of 10.3% during one year.
When looking specifically at the children actually participating in the study with at least one home
visit, the drop-out rate was only 4.8%, which compares favourably with the 47% drop-out rate in an
Australian study25
, where parents used a self-administered daily diary, and 11.9% in a Dutch
prospective birth cohort study141
. A recent Swiss birth cohort study assessing symptom frequency
by weekly telephone interviews had a drop-out rate of only 4.9% during one year78
.
37
Parents who gave birth to their first child were generally more eager to participate in the birth
cohort study because of the close contact to health professionals provided by monthly home visits.
However, we were aware of that and attempted to include an equal number of only children and
children with siblings to avoid selection bias. Likewise, families with very few resources were
underrepresented, as participation in the study required cooperation in the form of keeping a diary
and remembering the monthly appointments. In addition, some families might not like the idea of
letting a stranger into their private home. Overall, the cohort represents infants from a city area with
an overrepresentation of highly educated parents and a high rate of children being breastfed. The
cohort is therefore not representative for the whole country, but results may be referred to other city
areas in Denmark and to Western cities with a similar breastfeeding prevalence.
A sample size calculation showed that 200 children should be included in the study to be able to
detect differences in symptomatology between hMPV and RSV infections with an OR of 2.8.
However, this calculation was based on the assumption that all hMPV and RSV infections were
identified during the study period. Due to resource constraints and as 89% of anti-hMPV IgG-
positive children were also anti-RSV IgG-positive, we chose to analyse nasal swabs from the anti-
hMPV IgG-positive children only to detect the time of primary hMPV or RSV infection. In this
way, we identified 26.3% (10 out of 38) of the hMPV infections and 8.7% (15 out of 172) of the
RSV infections, which were too few to detect differences – if any - in symptomatology. The sample
size was sufficiently large to detect risk factors for hMPV and RSV infection with RR of 2.1 and
1.3 respectively, thereby reducing, however not eliminating, the possibility that findings may be due
to chance. In order to control confounding we used multiple logistic regression analysis for
estimating the magnitude of the association between the exposure and the outcome after adjusting
for a number of potential confounding factors. Furthermore possible effect modifications were
explored by generating interaction terms between some of the factors under investigation.
6.2. Specimen collection
In the excretion study, collection of nasal secretes was performed weekly and not daily, allowing
only a rough estimate of shedding duration of viral RNA. Parents collected urine and faeces
specimens from their child prior to our home visits, and occasionally, if samples were not ready at
our visit, they were sent by mail to the laboratory. These samples were frozen approximately two
days after collection, which might influence the stability of viral RNA in urine samples. However,
38
as S.T.A.R. buffer was added to the stool collection device, viral RNA in stool samples should not
be affected by this. As blood was obtained only from children who had a blood sample taken due to
their illness, no information on the presence of viral RNA in blood was available for half of the
included children. For collection of oral fluid the Oracol device was chosen, taken into account
acceptability, price, and quality145
.
Collection of sweat was done by using a device which is used routinously when testing children for
cyctic fibrosis. To avoid the risk of contamination, we washed the arm thoroughly with sterile water
prior to applying the pilocarpine disc and again prior to applying the spiral. We did not use ethanol
for washing as RSV has a lipid membrane that is easily destroyed by alcohol, which could also
affect the stability of RNA excreted in sweat110
. It is only possible to collect a relatively small
amount of sweat from young children, and freezing and thawing undiluted samples make the RNA
very sensitive to degradation. This made it difficult to reproduce our findings by RT-PCR and due
to lack of material we were not able to perform cell culture analyses and only succeeded in
sequencing one sample.
Nasal swab offers a sensitive sampling method for the detection of respiratory viruses in children.
RSV is an exception and it is detected more often in NPA than in nasal swabs when searched for by
ELISA, IFA, or viral culture56, 86, 130
. However, a recent study reports that when a sensitive
amplification method like RT-PCR is used for RSV analysis, nasal swabs have a sensitivity,
specificity, positive predictive value, and negative predictive value of 96%, 100%, 100%, and 91%,
respectively146
. The nasal swab specimens were chosen for surveillance of respiratory infections in
the birth cohort study, as they were sensitive in our own test of RSV in hospitalised infants, as they
are easy to collect, require no additional devices, and cause much less pain and distress than NPA86
.
As we collected nasal swabs at regular monthly intervals, nasal secretions from many ARTI
episodes were not available. We used this method to avoid the risk of bias and underreporting by
parents if they actively had to contact us for every respiratory episode, which has been the case in
other studies22
.
6.3. Laboratory methods used in the studies
In Papers I, II, and IV, real-time RT-PCR was used for RNA detection. Except for sweat and blood,
samples were extracted using the MagNA Pure LC Instrument. The PCR settings changed and
different protocols were used in the studies, but for each assay published primer sequences and
39
adequate controls were used and standard precautions taken to avoid cross and carry-over
contamination. Despite the fact that we used primers that only detected one subtype of hMPV in the
first study, the set up of each assay is superior to any other detection method of RSV and hMPV.
ELISA was used in Paper IV for detection of anti-hMPV and anti-RSV IgG in plasma from mothers
and children. Exact concentrations were determined for RSV IgG. We are not aware of the
existence of a standardised hMPV antigen with a known concentration, and the standard curve in
the hMPV ELISA was made from serial dilutions of a positive control without a known
concentration. The OD values did not allow us to determine an exact concentration of anti-hMPV
IgG, however, they could point in the direction of high or low IgG levels in a sample. As the
primary goal was a qualitative measurement of IgG in the samples, this is not a major limitation of
the assay. The inter- and intraassay variations of each assay were acceptable.
The Phadiatop Infant test was performed by Phadia ApS, Denmark. The diagnostic efficacy of the
test has been evaluated in studies of IgE sensitisation in young children with and without clinical
symptoms of atopic diseases5, 36
. In a prospective birth cohort study of unselected children, the
sensitivity of the Phadiatop Infant test was 96% among children characterised as IgE-sensitised
(positive skin prick test (SPT) and allergen-specific IgE) and 82% if children with either a positive
SPT or allergen-specific IgE were included. The specificity was 98%5.
6.4. Discussion of findings in each study
6.4.1. Paper I
The detection of hMPV in 2.9% of the children hospitalised with ARTI is in the lower end of what
has been reported in other studies of similar design2, 6, 107, 127
. As already mentioned, the use of
primer sequences detecting only one subtype of hMPV explains some of this lower prevalence.
Studies in which only samples tested negative for all known respiratory viruses were analysed for
hMPV naturally report higher frequencies of hMPV. Differences in patient types and sample
methodology may also explain differences in hMPV frequencies.
We detected hMPV-positive samples from end of December to mid March, with a peak in February.
As the study was designed to include NPAs collected during the winter season only, a seasonal
distribution covering a whole year was not determined. Other studies have found hMPV infection to
40
occur mainly during the winter season, although positive samples are also found during the summer
in contrast to RSV113, 149
.
Surprisingly, two thirds of the hMPV-infected children were discharged from hospital with a
diagnosis of asthmatic bronchitis. This was significantly more than for RSV-infected children. RSV
is in addition to rhinoviruses known to trigger asthma exacerbations in children and adults16
. The
relative role of hMPV in the induction of asthmatic phenotypes remains to be determined, but
findings from other studies indicate that hMPV plays an important role in the pathogenesis of
wheezing63, 92, 107
.
6.4.2. Paper II
We detected RSV RNA in faeces specimens from five children of whom four had diarrhoea. The
study is the first to detect RSV RNA in faeces. Recently, other respiratory viruses, such as
adenovirus84
, SARS-CoV26
, and human bocavirus144
, have been detected in stool. An explanation
for the finding of RSV RNA in stools might be that the children have swallowed respiratory
secretions containing RSV. The possibility that RSV replicates in the gastrointestinal tract seems
less likely. We did not perform viral culture analyses of the stools to confirm infectivity of the viral
RNA.
RSV and hMPV RNA were detected in sweat from four children who were under five weeks of age
or had a chronic lung disease, indicating that an immature or defective immune response facilitates
the spread of the virus from the upper respiratory tract. A short viraemic phase would allow the
virus to spread to the sweat glands to be excreted or to replicate. Other groups have detected RSV
and hMPV RNA in blood91, 115
, and recently rhinovirus viraemia has been described to occur in
normal children with a respiratory infection150
. No other groups have reported the detection or
search for respiratory viruses in sweat, but SARS-CoV has been detected in sweat glands of the
skin23
. The Ct values of the RT-PCR analyses of sweat were high, indicating that only few copies
of RNA were present in the samples. This makes high demands on the sensitivity of the detection
method, and low amounts of viral RNA are not likely to be detected by less sensitive methods like
IFA or ELISA. With the implementation of highly sensitive real-time RT-PCR systems in many
research laboratories around the world it will be interesting to see what future research will add to
our current understanding of viral excretion and transmission.
41
The median shedding durations of RSV and hMPV in nasal secretions were 11.5 and 5 days,
respectively. A previous study found that the shedding duration of RSV in hospitalised children
ranged from 1 to 21 days with a mean of 6.7 days measured by cell culture45
. Those of the children
with an LRTI shed virus for mean 8.4 days and those with a URTI for mean 1.5 days. No similar
studies have been published for hMPV. Our results are based on findings by RT-PCR, which may
be more sensitive than cell culture, explaining the longer shedding period in our study.
6.4.3. Paper III
Children in this study had one or more symptoms for 3.5 months during their first year of life. Not
many studies of similar design have been conducted. Previous Danish studies on this topic are either
cross-sectional, retrospective, focus mainly on older children, or follow children for a short period
of time53, 101, 102, 135
. Our findings are equal to the findings of a British longitudinal birth cohort
study of infants’ general symptoms59
, although we report higher prevalences of nasal symptoms,
fever, and cough. The reported prevalence of respiratory symptoms in our study is higher than for
most studies 25, 59, 78
, except studies from Greenland reporting respiratory symptoms on 41.6% of the
days of observation for children < 2 years of age69
.
We found that each child had on average 6.3 ARTI episodes per 365 days at risk. Including
episodes with simple rhinitis, each child had in total 9.7 respiratory episodes per 365 days at risk.
Most other studies report mean numbers of ARTI between 3.8 and 6.9 during the first year of life4, 7,
25, 60, 74, 81, 85, 96, although higher incidences are reported from Greenland
69 and Mozambique
114.
However, any direct comparisons between these studies and ours are difficult because of differences
in design, climate, demographics of the populations studied, definitions and classifications of
respiratory illness, the period required between episodes, and the methods of surveillance
employed.
The most substantial risk factors for respiratory symptoms in our study were increasing age, winter
season, and exposure to infections reflected by having siblings in a day nursery, number of
household members, living in small average rooms, and daycare attendance. The amount of disease
was relatively low in children under six months of age, after which a steep rise in respiratory
symptoms occurred. With increasing age several factors arise which influence on the child’s
42
susceptibility to infections, such as degradation of maternal antibodies, cessation of breastfeeding,
coming into more contact with others, and start at daycare centres at a time when the adaptive
immune system is still immature. Household size and size of residence were in our study only
associated with risk of simple rhinitis, indicating that crowding in the home, however important, is
mainly related to mild symptoms, while daycare attendance has a much higher impact on the
severity of respiratory illness. An important risk factor for respiratory symptoms was having
siblings aged 1-3 years. This is in accordance with other studies reporting that the number and age
of siblings predict the incidence of lower respiratory illness and wheezing in infancy25, 78, 82, 122
.
6.4.4. Paper IV
In this study, we found that risk factors for hMPV and RSV infection were different, as were risk
factors for mild and severe RSV infection. The study is the first to identify risk factors for primary
hMPV infection; presence of older siblings and being born in the spring. No other studies are
directly comparable with ours, but studies including children with underlying pathologies have
found prematurity, male gender, congenital heart disease and gastrointestinal reflux or aspiration to
be risk factors for hMPV hospitalisation113
. The finding that drying clothes inside protects against
hMPV infection is difficult to explain and may be due to chance.
We identified several factors, which were associated with RSV infection and hospitalisation. Some
risk factors were well known from previous studies (low GA, siblings, smoking, and lack of
breastfeeding15, 77, 103, 112
), while others were new to us: Increasing paternal age and wall-to-wall
carpeting. With increasing age, antibody levels decline, making the individual more susceptible to
new infections. An explanation could be that older fathers due to low levels of RSV antibodies
become infected and transmit the virus to the child. However, we did not collect plasma samples
from the fathers for antibody detection to confirm this theory.
The significance of carpeting is controversial with respect to allergic diseases; most studies report
carpeting to be associated with higher levels of house dust mites, thus aggravating the severity of
allergic asthma109, 139
. To our knowledge, the association between carpeting and RSV infection has
not been described previously. It is not known whether the survival of RSV is greater on carpets
than on smooth floors, but young children spend much time lying and crawling on the floor, and
transmission from surfaces contaminated with RSV-infected nasal secretions is possible45
. Maybe
parents are more inclined to place their child directly on a carpet rather than on a cold floor, and
43
maybe cleaning is easier achieved on the floor through rubbing. We found a non-significant
tendency of a protective effect of sleeping outside during the day. However, the sample size may
have been too small to reach statistical significance and further studies are warranted to confirm this
finding.
6.4.5. IgE-sensitisation
There is good evidence from clinical and experimental studies of RSV that LRTI may contribute to
early systemic sensitisation to common allergens104, 119
. Fifteen (7%) of the children in our study
had allergen-specific IgE detected in their blood at age 12 months, which is to be expected in a
cohort of healthy 1-year-old children61
. A positive test was associated with the presence of atopic
dermatitis, but not with hMPV or RSV infection, respiratory symptoms or diarrhoea, findings that
are in accordance with a recent Swedish study5. Low levels of specific IgE to food allergens are a
common phenomenon during early childhood and may have no clinical importance. However, the
presence of IgE antibodies against hen’s egg during early infancy has been shown to predict
respiratory allergy100
. A follow-up of this cohort, when the children reach school age, will show
whether these cases of low-grade IgE sensitisation at young ages are only temporary and without
clinical importance, or whether they have an impact on the development of allergic phenotypes at
older ages.
44
7. Conclusion and perspectives
Conclusion
The studies in the present thesis provide detailed data on the occurrence of ARTI in Danish infants
and verify that hMPV is present in young Danish children with ARTI, although less frequent than
RSV and with a tendency to a milder clinical course. hMPV and RSV have tropism for respiratory
epithelium cells, however, we detected viral RNA in faeces and sweat, indicating a systemic spread
of virus. Respiratory illness was associated with increasing age, winter season, household size,
daycare attendance, and having young siblings. Risk factors for early hMPV and RSV infection and
RSV hospitalisation were different; passive smoking and older siblings being determinants for a
severe outcome (i.e. hospitalisation).
Perspectives
Risk factors identified to be associated with ARTI in this study confirm the heterogeneity in the
factors as well as the limited potential for intervention. However, a reduction of illness might be
achieved by reducing transmission through improved hand hygiene practices in the homes and in
daycare institutions. Denmark still has a high prevalence of smokers compared with other European
countries, and public health policies must emphasize the harmful effects of passive smoking.
Further studies are warranted to define risk factors for severe hMPV infection. In addition, clinical
and virological studies are needed to elucidate the distribution of hMPV and RSV in different organ
systems and bodily fluids during infection and to determine whether viral RNA excreted in non-
respiratory secretions is infectious.
Complete elimination of the spread of RSV and hMPV is not possible, and prevention of disease by
vaccines may be the ultimate goal. Efforts are ongoing to generate an effective and safe vaccine
against RSV, and several candidate hMPV vaccines are under development. However, many
fundamental questions concerning the pathogenesis of hMPV disease and the host’s specific
immune response need to be answered before an effective vaccine strategy for hMPV can be
defined.
45
8. Summary
Acute respiratory tract infection (ARTI) in infants and young children is a significant public health
problem worldwide. Respiratory syncytial virus (RSV) is the leading cause of lower respiratory
tract infection in young children, but in many cases the etiological agent remains unknown. In 2001,
a hitherto unknown respiratory virus - human metapneumovirus (hMPV) - was identified in the
Netherlands. The discovery of hMPV forms the basis of the present thesis. The overall aim was to
study the clinical epidemiology of hMPV compared with RSV in hospitalised and non-hospitalised
children and to determine the frequency of acute respiratory symptoms in healthy infants. The thesis
includes four papers based on three studies:
In the first study, we examined the frequencies and clinical features of hMPV and RSV infections in
children hospitalised with ARTI. Nearly 400 nasopharyngeal aspirates from hospitalised children
were analysed for hMPV and RSV. The presence of hMPV among Danish children was confirmed
with the detection of 11 hMPV-positive samples and 190 RSV-positive samples. The symptoms
associated with hMPV and RSV infection were very much alike, however, more RSV-positive
children needed respiratory support. Asthmatic bronchitis was diagnosed more often in hMPV-
infected children.
In the second study, the excretion patterns of hMPV and RSV were investigated. During 3 weeks, 7
hMPV-positive and 38 RSV-positive children had 4 nasopharyngeal aspirates, saliva, faeces, and
urine samples taken in addition to 1 blood and 1 sweat sample. hMPV RNA was detected in saliva
and sweat. RSV RNA was found in saliva, faeces, and sweat. Children shedding viral RNA in sweat
were either younger than five weeks of age or had a chronic lung disease. Shedding durations for
hMPV and RSV were 5 and 11.5 days, respectively.
In the third study, a birth cohort of 228 healthy infants was followed for 12 months with monthly
home visits. Symptom diaries and nasal swabs were collected at every visit. Children had one or
more symptoms for 3.5 months during the first year of life, including runny nose for 2 months.
Each child had on average 6.3 episodes of ARTI (nasal discharge and ≥ 1 of the following
46
symptoms: cough, fever, wheezing, tachypnoea, malaise, or lost appetite) and 5.6 episodes of
simple rhinitis per 365 days at risk. Risk factors for respiratory symptoms were: increasing age,
winter season, household size, daycare attendance, and having siblings in a day nursery.
By age one year, 17.5% and 79% of the children had antibodies to hMPV and RSV, respectively.
Risk factors for acquired hMPV infection were: being born during the spring and having older
siblings. Risk factors for acquired RSV infection were: gestational age < 38 weeks, increasing
paternal age, and wall-to-wall carpeting, while being born during the spring protected against
infection. Risk factors for RSV hospitalisation were: older siblings and smoking in the household,
while exclusive breastfeeding for the first 14 days of life protected against hospitalisation.
In summary, these studies provide detailed data on the occurrence of ARTI in young Danish
children and verify that hMPV and RSV contribute to these infections. Symptoms associated with
hMPV and RSV infection are similar among both hospitalised and non-hospitalised children.
However, more RSV-infected children require respiratory support. Viral RNA is shed in faeces and
sweat indicating a systemic spread of virus. Risk factors identified to be associated with ARTI in
this study confirm the heterogeneity in the factors as well as the limited potential for intervention.
The burden of ARTI caused by viral pathogens is impressive and leaves no doubt that effective and
affordable vaccines are urgently needed.
47
9. Resumé på dansk
Luftvejsinfektioner hos små børn udgør et betydeligt globalt sundhedsproblem. Respiratorisk
syncytial virus (RSV) er den hyppigste årsag til nedre luftvejsinfektion hos mindre børn, men i
mange tilfælde forbliver det ætiologiske agens ukendt. I 2001 blev et nyt luftvejsvirus, human
metapneumovirus (hMPV), identificeret i Holland. Udgangspunktet for dette ph.d.-studie var
opdagelsen af hMPV. Hovedformålet med studiet var at belyse epidemiologien af hMPV
sammenlignet med RSV hos hospitaliserede og ikke-hospitaliserede børn og at undersøge
forekomsten af luftvejsinfektioner hos raske spædbørn. Afhandlingen indeholder 4 artikler baseret
på 3 studier:
I første studie blev forekomsten og de kliniske symptomer ved en hMPV og RSV infektion
undersøgt hos børn indlagt med akut luftvejsinfektion. Næsten 400 nasopharyngeal aspirater fra
indlagte børn blev undersøgt for hMPV og RSV. Forekomsten af hMPV hos danske børn blev
bekræftet med fundet af 11 hMPV-positive prøver og 190 RSV-positive prøver. Symptomerne ved
de 2 typer infektion var meget ens, men flere RSV-positive børn krævede respiratorisk støtte.
hMPV infektion var i højere grad end RSV infektion associeret med astmatisk bronkitis.
I andet studie blev udskillelsesmønstrene for hMPV og RSV undersøgt. Fra 7 hMPV-positive og 38
RSV-positive børn blev der over 3 uger opsamlet 4 nasopharyngeal aspirater, spyt, fæces- og
urinprøver samt en blod- og en svedprøve. hMPV RNA fandtes i spyt og sved. RSV RNA fandtes i
spyt, fæces og sved. Børn som udskilte viralt RNA i sved var enten yngre end 5 uger eller havde en
kronisk lungesygdom. Udskillelsesvarigheden for hMPV og RSV i nasal sekret var henholdsvis 5
og 11,5 dage.
I tredje studie fulgte vi en fødselskohorte på 228 raske børn med månedlige hjemmebesøg indtil 1-
årsalderen. Symptomdagbøger og næsepodninger blev indsamlet ved hvert besøg. Børnene havde et
eller flere symptomer i 3½ måned det første leveår, heraf forkølelse i 2 måneder. Hvert barn havde i
gennemsnit 6,3 episoder med luftvejsinfektion (løbenæse + hoste, feber, hvæsen, hurtig
vejrtrækning, pjevset eller nedsat appetit) og 5,6 episoder med løbenæse. Risikofaktorer for
48
luftvejsinfektion og løbenæse var: stigende alder, vintersæson, stor husstand, at gå i daginstitution
og at have søskende i vuggestue.
Henholdsvis 17,5% og 79% af børnene havde antistoffer mod hMPV og RSV ved 1-årsalderen som
tegn på erhvervet infektion. Risikofaktorer for hMPV infektion var: født i foråret og at have ældre
søskende. Risikofaktorer for RSV infektion var: født før fulde 38 uger, stigende fars alder og væg-
til-væg tæpper i boligen, mens at være født i foråret beskyttede mod smitte. Risikofaktorer for at
blive indlagt med RSV infektion var: ældre søskende og passiv rygning, mens fuld amning de første
14 dage beskyttede mod indlæggelse.
Sammenfattende giver studierne en detaljeret beskrivelse af forekomsten af luftvejsinfektion hos
danske småbørn, og viser at både hMPV og RSV forårsager en del af disse infektioner.
Symptomerne ved en hMPV og en RSV infektion er meget ens hos både indlagte og ikke-indlagte
børn, men børn med RSV infektion kræver oftere respiratorisk støtte. Viralt RNA udskilles i sved
og fæces tydende på en systemisk spredning af virus. Studierne bekræfter heterogeniteten af
risikofaktorer for luftvejsinfektion og de begrænsede muligheder for intervention. Sygdomsbyrden
ved virale luftvejsinfektioner er betydelig og understreger behovet for effektive og tilgængelige
vacciner.
49
10. References
1. Abels S, Nadal D, Stroehle A, Bossart W. Reliable detection of respiratory syncytial virus
infection in children for adequate hospital infection control management. J Clin Microbiol
2001;39(9):3135-3139.
2. Al-Sonboli N, Hart CA, Al-Aeryani A, Banajeh SM, Al-Aghbari N, Dove W, Cuevas LE.
Respiratory syncytial virus and human metapneumovirus in children with acute respiratory
infections in Yemen. Pediatr Infect Dis J 2005;24(8):734-736.
3. Alvarez R, Harrod KS, Shieh WJ, Zaki S, Tripp RA. Human metapneumovirus persists in
BALB/c mice despite the presence of neutralizing antibodies. J Virol 2004;78(24):14003-
14011.
4. Badger GF, Dingle JH, Feller AE, Hodges RG, Jordan WS, Jr., Rammelkamp CH, Jr. A
study of illness in a group of Cleveland families. II. Incidence of the common respiratory
diseases. Am J Hyg 1953;58(1):31-40.
5. Ballardini N, Nilsson C, Nilsson M, Lilja G. ImmunoCAP Phadiatop Infant - a new blood
test for detecting IgE sensitisation in children at 2 years of age. Allergy 2006;61(3):337-343.
6. Boivin G, De Serras G, Côté S, Gilca R, Abed Y, Rochette L, Bergeron MG, Déry P.
Human metapneumovirus infections in hospitalized children. Emerg Infect Dis
2003;9(6):634-640.
7. Borrero I, Fajardo L, Bedoya A, Zea A, Carmona F, de Borrero MF. Acute respiratory tract
infections among a birth cohort of children from Cali, Colombia, who were studied through
17 months of age. Rev Infect Dis 1990;12 Suppl 8:S950-S956.
8. Bosis S, Esposito S, Niesters HG, Crovari P, Osterhaus AD, Principi N. Impact of human
metapneumovirus in childhood: comparison with respiratory syncytial virus and influenza
viruses. J Med Virol 2005;75(1):101-104.
9. Bouscambert-Duchamp M, Lina B, Trompette A, Moret H, Motte J, Andreoletti L.
Detection of human metapneumovirus RNA sequences in nasopharyngeal aspirates of young
French children with acute bronchiolitis by real-time reverse transcriptase PCR and
phylogenetic analysis. J Clin Microbiol 2005;43(3):1411-1414.
10. Bulut Y, Guven M, Otlu B, Yenisehirli G, Aladag I, Eyibilen A, Dogru S. Acute otitis media
and respiratory viruses. Eur J Pediatr 2007;166(3):223-228.
11. Butz A. Use of health diaries in pediatric research. J Pediatr Health Care 2004;18(5):262-
263.
50
12. Carabin H, Gyorkos TW, Soto JC, Joseph L, Payment P, Collet JP. Effectiveness of a
training program in reducing infections in toddlers attending day care centers. Epidemiology
1999;10(3):219-227.
13. Casiano-Colon AE, Hulbert BB, Mayer TK, Walsh EE, Falsey AR. Lack of sensitivity of
rapid antigen tests for the diagnosis of respiratory syncytial virus infection in adults. J Clin
Virol 2003;28(2):169-174.
14. Chanock R, Roizman B, Myers R. Recovery from infants with respiratory illness of a virus
related to chimpanzee coryza agent (CCA). I. Isolation, properties and characterization. Am
J Hyg 1957;66(3):281-290.
15. Cilla G, Sarasua A, Montes M, Arostegui N, Vicente D, Perez-Yarza E, Perez-Trallero E.
Risk factors for hospitalization due to respiratory syncytial virus infection among infants in
the Basque Country, Spain. Epidemiol Infect 2006;134(3):506-513.
16. Cohen L, Castro M. The role of viral respiratory infections in the pathogenesis and
exacerbation of asthma. Semin Respir Infect 2003;18(1):3-8.
17. Collins PL, Chanock RM, Murphy BR. Respiratory syncytial virus. In: Knipe DM, Howley
PM, eds. Fields virology. Philadelphia: Lippincott Williams & Wilkins, 2001:1443-1485.
18. Crowe JE, Jr., Williams JV. Immunology of viral respiratory tract infection in infancy.
Paediatr Respir Rev 2003;4(2):112-119.
19. Cushing AH, Samet JM, Lambert WE, Skipper BJ, Hunt WC, Young SA, McLaren LC.
Breastfeeding reduces risk of respiratory illness in infants. Am J Epidemiol
1998;147(9):863-870.
20. Danmarks statistik. Befolkningens uddannelse og beskæftigelse (Danish). www.dst.dk /
September 2007.
21. Darville T, Yamauchi T. Respiratory syncytial virus. Pediatr Rev 1998;19(2):55-61.
22. de Waal L, Koopman LP, van Benten IJ, Brandenburg AH, Mulder PG, de Swart RL,
Fokkens WJ, Neijens HJ, Osterhaus AD. Moderate local and systemic respiratory syncytial
virus-specific T-cell responses upon mild or subclinical RSV infection. J Med Virol
2003;70(2):309-318.
23. Ding Y, He L, Zhang Q, Huang Z, Che X, Hou J, Wang H, Shen H, Qiu L, Li Z, Geng J, Cai
J, Han H, Li X, Kang W, Weng D, Liang P, Jiang S. Organ distribution of severe acute
respiratory syndrome (SARS) associated coronavirus (SARS-CoV) in SARS patients:
implications for pathogenesis and virus transmission pathways. J Pathol 2004;203(2):622-
630.
24. Døllner H, Risnes K, Radtke A, Nordbø SA. Outbreak of human metapneumovirus infection
in Norwegian children. Pediatr Infect Dis J 2004;23(5):436-440.
51
25. Douglas RM, Woodward A, Miles H, Buetow S, Morris D. A prospective study of
proneness to acute respiratory illness in the first two years of life. Int J Epidemiol
1994;23(4):818-826.
26. Drosten C, Gunther S, Preiser W, van der Werf S, Brodt HR, Becker S, Rabenau H, Panning
M, Kolesnikova L, Fouchier RA, Berger A, Burguiere AM, Cinatl J, Eickmann M, Escriou
N, Grywna K, Kramme S, Manuguerra JC, Muller S, Rickerts V, Sturmer M, Vieth S, Klenk
HD, Osterhaus AD, Schmitz H, Doerr HW. Identification of a Novel Coronavirus in Patients
with Severe Acute Respiratory Syndrome. N Engl J Med 2003;348(20):1967-1976.
27. Ebihara T, Endo R, Kikuta H, Ishiguro N, Ishiko H, Hara M, Takahashi Y, Kobayashi K.
Human metapneumovirus infection in Japanese children. J Clin Microbiol 2004;42(1):126-
132.
28. Ebihara T, Endo R, Kikuta H, Ishiguro N, Yoshioka M, Ma X, Kobayashi K.
Seroprevalence of human metapneumovirus in Japan. J Med Virol 2003;70(2):281-283.
29. Ebihara T, Endo R, Ma X, Ishiguro N, Kikuta H. Detection of human metapneumovirus
antigens in nasopharyngeal secretions by an immunofluorescent-antibody test. J Clin
Microbiol 2005;43(3):1138-1141.
30. Eisenhut M. Extrapulmonary manifestations of severe respiratory syncytial virus infection--
a systematic review. Crit Care 2006;10(4):R107.
31. Esper F, Boucher D, Weibel C, Martinello RA, Kahn JS. Human metapneumovirus infection
in the United States: clinical manifestations associated with a newly emerging respiratory
infection in children. Pediatrics 2003;111(6 Pt 1):1407-1410.
32. Esper F, Martinello RA, Boucher D, Weibel C, Ferguson D, Landry ML, Kahn JS. A 1-year
experience with human metapneumovirus in children aged <5 years. J Infect Dis
2004;189(8):1388-1396.
33. Falsey AR, Criddle MC, Walsh EE. Detection of respiratory syncytial virus and human
metapneumovirus by reverse transcription polymerase chain reaction in adults with and
without respiratory illness. J Clin Virol 2006;35(1):46-50.
34. Falsey AR, Formica MA, Walsh EE. Diagnosis of respiratory syncytial virus infection:
comparison of reverse transcription-PCR to viral culture and serology in adults with
respiratory illness. J Clin Microbiol 2002;40(3):817-820.
35. Falsey AR, Hennessey PA, Formica MA, Cox C, Walsh EE. Respiratory Syncytial Virus
Infection in Elderly and High-Risk Adults. The New England Journal of Medicine
2005;352(17):1749-1759.
36. Fiocchi A, Besana R, Ryden AC, Terracciano L, Andreotti M, Arrigoni S, Martelli A.
Differential diagnosis of IgE-mediated allergy in young children with wheezing or eczema
symptoms using a single blood test. Ann Allergy Asthma Immunol 2004;93(4):328-333.
37. Fixler DE. Respiratory syncytial virus infection in children with congenital heart disease: a
review. Pediatr Cardiol 1996;17(3):163-168.
52
38. Forster J, Tacke U, Krebs H, Streckert HJ, Werchau H, Bergmann RL, Schulz J, Lau S,
Wahn U. Respiratory syncytial virus infection: its role in aeroallergen sensitization during
the first two years of life. Pediatr Allergy Immunol 1996;7(2):55-60.
39. Freymouth F, Vabret A, Legrand L, Eterradossi N, Lafay-Delaire F, Brouard J, Guillois B.
Presence of the new human metapneumovirus in French children with bronchiolitis. Pediatr
Infect Dis J 2003;22(1):92-94.
40. García-García ML, Calvo C, Martín F, Pérez-Breña P, Acosta B, Casas I. Human
metapneumovirus infections in hospitalised infants in Spain. Arch Dis Child
2006;91(4):290-295.
41. Glaser CA, Honarmand S, Anderson LJ, Schnurr DP, Forghani B, Cossen CK, Schuster FL,
Christie LJ, Tureen JH. Beyond viruses: clinical profiles and etiologies associated with
encephalitis. Clin Infect Dis 2006;43(12):1565-1577.
42. Glezen WP, Taber LH, Frank AL, Kasel JA. Risk of primary infection and reinfection with
respiratory syncytial virus. Am J Dis Child 1986;140(6):543-546.
43. Graham NM. The epidemiology of acute respiratory infections in children and adults: a
global perspective. Epidemiol Rev 1990;12:149-178.
44. Groth C, von Linstow ML, Høgh B. Behandling af bronchiolitis hos børn [Treatment of
bronchiolitis in children] (Danish). Ugeskr Laeger 2007;In press.
45. Hall CB. The shedding and spreading of respiratory syncytial virus. Pediatr Res 1977;11(3
Pt 2):236-239.
46. Hall CB, Douglas RG, Jr. Modes of transmission of respiratory syncytial virus. J Pediatr
1981;99(1):100-103.
47. Hall CB, Douglas RG, Jr., Geiman JM. Respiratory syncytial virus infections in infants:
quantitation and duration of shedding. J Pediatr 1976;89(1):11-15.
48. Hall CB, Douglas RG, Jr., Geiman JM. Possible transmission by fomites of respiratory
syncytial virus. J Infect Dis 1980;141(1):98-102.
49. Hall CB, Geiman JM, Biggar R, Kotok DI, Hogan PM, Douglas GR, Jr. Respiratory
syncytial virus infections within families. N Engl J Med 1976;294(8):414-419.
50. Hall CB, Powell KR, MacDonald NE, Gala CL, Menegus ME, Suffin SC, Cohen HJ.
Respiratory syncytial viral infection in children with compromised immune function. N
Engl J Med 1986;315(2):77-81.
51. Hall CB, Walsh EE, Long CE, Schnabel KC. Immunity to and frequency of reinfection with
respiratory syncytial virus. J Infect Dis 1991;163(4):693-698.
53
52. Hall CB, Walsh EE, Schnabel KC, Long CE, McConnochie KM, Hildreth SW, Anderson
LJ. Occurrence of groups A and B of respiratory syncytial virus over 15 years: associated
epidemiologic and clinical characteristics in hospitalized and ambulatory children. J Infect
Dis 1990;162(6):1283-1290.
53. Hansen BW. Acute illnesses in children. A description and analysis of the cumulative
incidence proportion. Scand J Prim Health Care 1993;11(3):202-206.
54. Hansen EJ. Socialgrupper i Danmark (Danish). Socialforskningsinstituttet. Studie 48. 1984.
55. Hayes EB, Hurwitz ES, Schonberger LB, Anderson LJ. Respiratory syncytial virus outbreak
on American Samoa. Evaluation of risk factors. Am J Dis Child 1989;143(3):316-321.
56. Heikkinen T, Marttila J, Salmi AA, Ruuskanen O. Nasal swab versus nasopharyngeal
aspirate for isolation of respiratory viruses. J Clin Microbiol 2002;40(11):4337-4339.
57. Henderson FW, Collier AM, Clyde WA, Jr., Denny FW. Respiratory-syncytial-virus
infections, reinfections and immunity. A prospective, longitudinal study in young children.
N Engl J Med 1979;300(10):530-534.
58. Holberg CJ, Wright AL, Martinez FD, Ray CG, Taussig LM, Lebowitz MD. Risk factors for
respiratory syncytial virus-associated lower respiratory illnesses in the first year of life. Am
J Epidemiol 1991;133(11):1135-1151.
59. Holme CO. Incidence and prevalence of non-specific symptoms and behavioural changes in
infants under the age of two years. Br J Gen Pract 1995;45(391):65-69.
60. Hortal M, Benitez A, Contera M, Etorena P, Montano A, Meny M. A community-based
study of acute respiratory tract infections in children in Uruguay. Rev Infect Dis 1990;12
Suppl 8:S966-S973.
61. Høst A, Halken S. The role of allergy in childhood asthma. Allergy 2000;55(7):600-608.
62. Isaacs D, Dickson H, O'Callaghan C, Sheaves R, Winter A, Moxon ER. Handwashing and
cohorting in prevention of hospital acquired infections with respiratory syncytial virus. Arch
Dis Child 1991;66(2):227-231.
63. Jartti T, van den Hoogen BG, Garofalo RP, Osterhaus AD, Ruuskanen O. Metapneumovirus
and acute wheezing in children. Lancet 2002;360(9343):1393-1394.
64. Juntti H, Kokkonen J, Dunder T, Renko M, Niinimaki A, Uhari M. Association of an early
respiratory syncytial virus infection and atopic allergy. Allergy 2003;58(9):878-884.
65. Kahn JS. Human metapneumovirus: a newly emerging respiratory pathogen. Curr Opin
Infect Dis 2003;16(3):255-258.
66. Kaida A, Iritani N, Kubo H, Shiomi M, Kohdera U, Murakami T. Seasonal distribution and
phylogenetic analysis of human metapneumovirus among children in Osaka City, Japan. J
Clin Virol 2006;35(4):394-399.
54
67. Kamper-Jørgensen M, Wohlfahrt J, Simonsen J, Grønbæk M, Benn CS. Population-based
study of the impact of childcare attendance on hospitalizations for acute respiratory
infections. Pediatrics 2006;118(4):1439-1446.
68. Koch A, Mølbak K, Homøe P, Sørensen P, Hjuler T, Olesen ME, Pejl J, Pedersen FK, Olsen
OR, Melbye M. Risk factors for acute respiratory tract infections in young Greenlandic
children. Am J Epidemiol 2003;158(4):374-384.
69. Koch A, Sørensen P, Homøe P, Mølbak K, Pedersen FK, Mortensen T, Elberling H, Eriksen
AM, Olsen OR, Melbye M. Population-based study of acute respiratory infections in
children, Greenland. Emerg Infect Dis 2002;8(6):586-593.
70. Konig B, Konig W, Arnold R, Werchau H, Ihorst G, Forster J. Prospective study of human
metapneumovirus infection in children less than 3 years of age. J Clin Microbiol
2004;42(10):4632-4635.
71. Korppi M, Piippo-Savolainen E, Korhonen K, Remes S. Respiratory morbidity 20 years
after RSV infection in infancy. Pediatr Pulmonol 2004;38(2):155-160.
72. Kristensen K, Dahm T, Frederiksen PS, Ibsen J, Iyore E, Jensen AM, Kjær BB, Olofsson K,
Pedersen P, Poulsen S. Epidemiology of respiratory syncytial virus infection requiring
hospitalization in East Denmark. Pediatr Infect Dis J 1998;17(11):996-1000.
73. Kuiken T, van den Hoogen BG, van Riel DA, Laman JD, van Amerongen G, Sprong L,
Fouchier RA, Osterhaus AD. Experimental human metapneumovirus infection of
cynomolgus macaques (Macaca fascicularis) results in virus replication in ciliated epithelial
cells and pneumocytes with associated lesions throughout the respiratory tract. Am J Pathol
2004;164(6):1893-1900.
74. Kusel MM, de Klerk NH, Holt PG, Kebadze T, Johnston SL, Sly PD. Role of respiratory
viruses in acute upper and lower respiratory tract illness in the first year of life: a birth
cohort study. Pediatr Infect Dis J 2006;25(8):680-686.
75. Kuypers J, Wright N, Corey L, Morrow R. Detection and quantification of human
metapneumovirus in pediatric specimens by real-time RT-PCR. J Clin Virol
2005;33(4):299-305.
76. Kuypers J, Wright N, Morrow R. Evaluation of quantitative and type-specific real-time RT-
PCR assays for detection of respiratory syncytial virus in respiratory specimens from
children. J Clin Virol 2004;31(2):123-129.
77. Lanari M, Giovannini M, Giuffre L, Marini A, Rondini G, Rossi GA, Merolla R, Zuccotti
GV, Salvioli GP. Prevalence of respiratory syncytial virus infection in Italian infants
hospitalized for acute lower respiratory tract infections, and association between respiratory
syncytial virus infection risk factors and disease severity. Pediatr Pulmonol 2002;33(6):458-
465.
78. Latzin P, Frey U, Roiha HL, Baldwin DN, Regamey N, Strippoli MP, Zwahlen M, Kuehni
CE. Prospectively assessed incidence, severity, and determinants of respiratory symptoms in
the first year of life. Pediatr Pulmonol 2007;42(1):41-50.
55
79. Lazar I, Weibel C, Dziura J, Ferguson D, Landry ML, Kahn JS. Human metapneumovirus
and severity of respiratory syncytial virus disease. Emerg Infect Dis 2004;10(7):1318-1320.
80. Leader S, Kohlhase K. Respiratory syncytial virus-coded pediatric hospitalizations, 1997 to
1999. Pediatr Infect Dis J 2002;21(7):629-632.
81. Leder K, Sinclair MI, Mitakakis TZ, Hellard ME, Forbes A. A community-based study of
respiratory episodes in Melbourne, Australia. Aust N Z J Public Health 2003;27(4):399-404.
82. Leeder SR, Corkhill R, Irwig LM, Holland WW, Colley JR. Influence of family factors on
the incidence of lower respiratory illness during the first year of life. Br J Prev Soc Med
1976;30(4):203-212.
83. Leung J, Esper F, Weibel C, Kahn JS. Seroepidemiology of human metapneumovirus
(hMPV) on the basis of a novel enzyme-linked immunosorbent assay utilizing hMPV fusion
protein expressed in recombinant vesicular stomatitis virus. J Clin Microbiol
2005;43(3):1213-1219.
84. Lew JF, Glass RI, Petric M, LeBaron CW, Hammond GW, Miller SE, Robinson C, Boutilier
J, Riepenhoff-Talty M, Payne CM. Six-year retrospective surveillance of gastroenteritis
viruses identified at ten electron microscopy centers in the United States and Canada.
Pediatr Infect Dis J 1990;9(10):709-714.
85. López Bravo IM, Sepúlveda H, Valdés I. Acute respiratory illnesses in the first 18 months of
life. Rev Panam Salud Publica 1997;1(1):9-17.
86. Macfarlane P, Denham J, Assous J, Hughes C. RSV testing in bronchiolitis: which nasal
sampling method is best? Arch Dis Child 2005;90(6):634-635.
87. Mackay IM, Jacob KC, Woolhouse D, Waller K, Syrmis MW, Whiley DM, Siebert DJ,
Nissen M, Sloots TP. Molecular assays for detection of human metapneumovirus. J Clin
Microbiol 2003;41(1):100-105.
88. Madhi SA, Klugman KP. A role for Streptococcus pneumoniae in virus-associated
pneumonia. Nat Med 2004;10(8):811-813.
89. Madhi SA, Ludewick H, Kuwanda L, Niekerk N, Cutland C, Little T, Klugman KP.
Pneumococcal coinfection with human metapneumovirus. J Infect Dis 2006;193(9):1236-
1243.
90. Maertzdorf J, Wang CK, Brown JB, Quinto JD, Chu M, de Graaf M, van den Hoogen BG,
Spaete R, Osterhaus AD, Fouchier RA. Real-time reverse transcriptase PCR assay for
detection of human metapneumoviruses from all known genetic lineages. J Clin Microbiol
2004;42(3):981-986.
91. Maggi F, Pifferi M, Vatteroni M, Fornai C, Tempestini E, Anzilotti S, Lanini L, Andreoli E,
Ragazzo V, Pistello M, Specter S, Bendinelli M. Human metapneumovirus associated with
respiratory tract infections in a 3-year study of nasal swabs from infants in Italy. J Clin
Microbiol 2003;41(7):2987-2991.
56
92. Manoha C, Espinosa S, Aho SL, Huet F, Pothier P. Epidemiological and clinical features of
hMPV, RSV and RVs infections in young children. J Clin Virol 2007;38(3):221-226.
93. McAdam AJ, Hasenbein ME, Feldman HA, Cole SE, Offermann JT, Riley AM, Lieu TA.
Human metapneumovirus in children tested at a tertiary-care hospital. J Infect Dis
2004;190(1):20-26.
94. McIntosh K. Respiratory Syncytial Virus Infections in Infants and Children: Diagnosis and
Treatment. Pediatrics in Review 1987;9(6):191-196.
95. Mentel R, Wegner U, Bruns R, Gurtler L. Real-time PCR to improve the diagnosis of
respiratory syncytial virus infection. J Med Microbiol 2003;52(Pt 10):893-896.
96. Monto AS, Ullman BM. Acute respiratory illness in an American community. The
Tecumseh study. JAMA 1974;227(2):164-169.
97. Mullins JA, Erdman DD, Weinberg GA, Edwards K, Hall CB, Walker FJ, Iwane M,
Anderson LJ. Human metapneumovirus infection among children hospitalized with acute
respiratory illness. Emerg Infect Dis 2004;10(4):700-705.
98. Murphy BR, Alling DW, Snyder MH, Walsh EE, Prince GA, Chanock RM, Hemming VG,
Rodriguez WJ, Kim HW, Graham BS. Effect of age and preexisting antibody on serum
antibody response of infants and children to the F and G glycoproteins during respiratory
syncytial virus infection. J Clin Microbiol 1986;24(5):894-898.
99. Navas L, Wang E, de Carvalho V, Robinson J. Improved outcome of respiratory syncytial
virus infection in a high-risk hospitalized population of Canadian children. Pediatric
Investigators Collaborative Network on Infections in Canada. J Pediatr 1992;121(3):348-
354.
100. Nickel R, Kulig M, Forster J, Bergmann R, Bauer CP, Lau S, Guggenmoos-Holzmann I,
Wahn U. Sensitization to hen's egg at the age of twelve months is predictive for allergic
sensitization to common indoor and outdoor allergens at the age of three years. J Allergy
Clin Immunol 1997;99(5):613-617.
101. Nielsen AM, Koefoed BG, Møller R, Laursen B. [Prevalence rates of recent illnesses in
Danish children, 1994 and 2000] (Danish). Ugeskr Laeger 2006;168(4):373-378.
102. Nielsen AM, Rasmussen S, Christoffersen MN. [Morbidity of Danish infants during their
first months of life. Incidence and risk factors] (Danish). Ugeskr Laeger 2002;164(48):5644-
5648.
103. Nielsen HE, Siersma V, Andersen S, Gahrn-Hansen B, Mordhorst CH, Nørgaard-Pedersen
B, Røder B, Sørensen TL, Temme R, Vestergaard BF. Respiratory syncytial virus infection-
risk factors for hospital admission: a case-control study. Acta Paediatr 2003;92(11):1314-
1321.
104. Ogra PL. Respiratory syncytial virus: the virus, the disease and the immune response.
Paediatr Respir Rev 2004;5 Suppl A:S119-S126.
57
105. Ordás J, Boga JA, Alvarez-Argüelles M, Villa L, Rodriguez-Dehli C, de Oña M, Rodríguez
J, Melón S. Role of metapneumovirus in viral respiratory infections in young children. J
Clin Microbiol 2006;44(8):2739-2742.
106. Patel JA, Nguyen DT, Revai K, Chonmaitree T. Role of respiratory syncytial virus in acute
otitis media: implications for vaccine development. Vaccine 2007;25(9):1683-1689.
107. Peiris JS, Tang WH, Chan KH, Khong PL, Guan Y, Lau YL, Chiu SS. Children with
respiratory disease associated with metapneumovirus in Hong Kong. Emerg Infect Dis
2003;9(6):628-633.
108. Percivalle E, Sarasini A, Visai L, Revello MG, Gerna G. Rapid detection of human
metapneumovirus strains in nasopharyngeal aspirates and shell vial cultures by monoclonal
antibodies. J Clin Microbiol 2005;43(7):3443-3446.
109. Perry TT, Wood RA, Matsui EC, Curtin-Brosnan J, Rand C, Eggleston PA. Room-specific
characteristics of suburban homes as predictors of indoor allergen concentrations. Ann
Allergy Asthma Immunol 2006;97(5):628-635.
110. Platt J, Bucknall RA. The disinfection of respiratory syncytial virus by isopropanol and a
chlorhexidine-detergent handwash. J Hosp Infect 1985;6(1):89-94.
111. Pullan CR, Hey EN. Wheezing, asthma, and pulmonary dysfunction 10 years after infection
with respiratory syncytial virus in infancy. Br Med J (Clin Res Ed) 1982;284(6330):1665-
1669.
112. Pullan CR, Toms GL, Martin AJ, Gardner PS, Webb JK, Appleton DR. Breast-feeding and
respiratory syncytial virus infection. Br Med J 1980;281(6247):1034-1036.
113. Robinson JL, Lee BE, Bastien N, Li Y. Seasonality and clinical features of human
metapneumovirus infection in children in Northern Alberta. J Med Virol 2005;76(1):98-105.
114. Roca A, Quinto L, Saute F, Thompson R, Aponte JJ, Alonso PL. Community incidences of
respiratory infections in an actively followed cohort of children <1 year of age in Manhica, a
rural area of southern Mozambique. Trop Med Int Health 2006;11(3):373-380.
115. Rohwedder A, Keminer O, Forster J, Schneider K, Schneider E, Werchau H. Detection of
respiratory syncytial virus RNA in blood of neonates by polymerase chain reaction. J Med
Virol 1998;54(4):320-327.
116. Rossi GA, Medici MC, Arcangeletti MC, Lanari M, Merolla R, Paparatti UD, Silvestri M,
Pistorio A, Chezzi C. Risk factors for severe RSV-induced lower respiratory tract infection
over four consecutive epidemics. Eur J Pediatr 2007.
117. Ruuskanen O. Respiratory syncytial virus - is it preventable? J Hosp Infect 1995;30
Suppl:494-497.
58
118. Sandora TJ, Taveras EM, Shih MC, Resnick EA, Lee GM, Ross-Degnan D, Goldmann DA.
A randomized, controlled trial of a multifaceted intervention including alcohol-based hand
sanitizer and hand-hygiene education to reduce illness transmission in the home. Pediatrics
2005;116(3):587-594.
119. Schauer U, Hoffjan S, Bittscheidt J, Kochling A, Hemmis S, Bongartz S, Stephan V. RSV
bronchiolitis and risk of wheeze and allergic sensitisation in the first year of life. Eur Respir
J 2002;20(5):1277-1283.
120. Schildgen O, Glatzel T, Geikowski T, Scheibner B, Matz B, Bindl L, Born M, Viazov S,
Wilkesmann A, Knopfle G, Roggendorf M, Simon A. Human metapneumovirus RNA in
encephalitis patient. Emerg Infect Dis 2005;11(3):467-470.
121. Semple MG, Cowell A, Dove W, Greensill J, McNamara PS, Halfhide C, Shears P, Smyth
RL, Hart CA. Dual infection of infants by human metapneumovirus and human respiratory
syncytial virus is strongly associated with severe bronchiolitis. J Infect Dis
2005;191(3):382-386.
122. Sherriff A, Peters TJ, Henderson J, Strachan D. Risk factor associations with wheezing
patterns in children followed longitudinally from birth to 3(1/2) years. Int J Epidemiol
2001;30(6):1473-1484.
123. Sigurs N, Bjarnason R, Sigurbergsson F, Kjellman B. Respiratory syncytial virus
bronchiolitis in infancy is an important risk factor for asthma and allergy at age 7. Am J
Respir Crit Care Med 2000;161(5):1501-1507.
124. Sigurs N, Bjarnason R, Sigurbergsson F, Kjellman B, Bjorksten B. Asthma and
immunoglobulin E antibodies after respiratory syncytial virus bronchiolitis: a prospective
cohort study with matched controls. Pediatrics 1995;95(4):500-505.
125. Sigurs N, Gustafsson PM, Bjarnason R, Lundberg F, Schmidt S, Sigurbergsson F, Kjellman
B. Severe respiratory syncytial virus bronchiolitis in infancy and asthma and allergy at age
13. Am J Respir Crit Care Med 2005;171(2):137-141.
126. Simoes EA. Respiratory syncytial virus infection. Lancet 1999;354(9181):847-852.
127. Sloots TP, Mackay IM, Bialasiewicz S, Jacob KC, McQueen E, Harnett GB, Siebert DJ,
Masters BI, Young PR, Nissen MD. Human metapneumovirus, Australia, 2001-2004.
Emerg Infect Dis 2006;12(8):1263-1266.
128. Staat MA. Respiratory syncytial virus infections in children. Semin Respir Infect
2002;17(1):15-20.
129. Stein RT, Sherrill D, Morgan WJ, Holberg CJ, Halonen M, Taussig LM, Wright AL,
Martinez FD. Respiratory syncytial virus in early life and risk of wheeze and allergy by age
13 years. Lancet 1999;354(9178):541-545.
59
130. Stensballe LG, Trautner S, Kofoed PE, Nante E, Hedegaard K, Jensen IP, Aaby P.
Comparison of nasopharyngeal aspirate and nasal swab specimens for detection of
respiratory syncytial virus in different settings in a developing country. Trop Med Int Health
2002;7(4):317-321.
131. Stensballe LG, Kristensen K, Simoes EA, Jensen H, Nielsen J, Benn CS, Aaby P, for the
Danish RSV Data Network. Atopic Disposition, Wheezing, and Subsequent Respiratory
Syncytial Virus Hospitalization in Danish Children Younger Than 18 Months: A Nested
Case-Control Study. Pediatrics 2006;118(5):e1360-e1368.
132. Strangert K. Respiratory illness in preschool children with different forms of day care.
Pediatrics 1976;57(2):191-196.
133. Sundhedsstyrelsen. Antallet af rygere i Danmark falder fortsat (Danish). www.sst.dk /
September 2007.
134. Trefny P, Stricker T, Baerlocher C, Sennhauser FH. Family history of atopy and clinical
course of RSV infection in ambulatory and hospitalized infants. Pediatr Pulmonol
2000;30(4):302-306.
135. Uldall P. Spæd- og småbørns almindelige sygelighed - forekomst og sociale konsekvenser
[Acute illness in preschool children] (Danish). Thesis 1986;(Publ. 18).
136. van den Hoogen BG, de Jong JC, Groen J, Kuiken T, de Groot R, Fouchier RA, Osterhaus
AD. A newly discovered human pneumovirus isolated from young children with respiratory
tract disease. Nat Med 2001;7(6):719-724.
137. van den Hoogen BG, Osterhaus DM, Fouchier RA. Clinical impact and diagnosis of human
metapneumovirus infection. Pediatr Infect Dis J 2004;23(1 Suppl):S25-S32.
138. van den Hoogen BG, van Doornum GJ, Fockens JC, Cornelissen JJ, Beyer WE, de Groot R,
Osterhaus AD, Fouchier RA. Prevalence and clinical symptoms of human metapneumovirus
infection in hospitalized patients. J Infect Dis 2003;188(10):1571-1577.
139. van Strien RT, Verhoeff AP, Brunekreef B, van Wijnen JH. Mite antigen in house dust:
relationship with different housing characteristics in The Netherlands. Clin Exp Allergy
1994;24(9):843-853.
140. van Woensel JB, Bos AP, Lutter R, Rossen JW, Schuurman R. Absence of human
metapneumovirus co-infection in cases of severe respiratory syncytial virus infection.
Pediatr Pulmonol 2006;41(9):872-874.
141. van Benten I, Koopman L, Niesters B, Hop W, van Middelkoop B, de Waal L, van Drunen
K, Osterhaus AD, Neijens H, Fokkens W. Predominance of rhinovirus in the nose of
symptomatic and asymptomatic infants. Pediatr Allergy Immunol 2003;14(5):363-370.
142. Verbrugge LM. Health diaries. Med Care 1980;18(1):73-95.
60
143. Viazov S, Ratjen F, Scheidhauer R, Fiedler M, Roggendorf M. High prevalence of human
metapneumovirus infection in young children and genetic heterogeneity of the viral isolates.
J Clin Microbiol 2003;41(7):3043-3045.
144. Vicente D, Cilla G, Montes M, Perez-Yarza EG, Perez-Trallero E. Human bocavirus, a
respiratory and enteric virus. Emerg Infect Dis 2007;13(4):636-637.
145. Vyse AJ, Cohen BJ, Ramsay ME. A comparison of oral fluid collection devices for use in
the surveillance of virus diseases in children. Public Health 2001;115(3):201-207.
146. Waris M, Heikkinen T, Osterback R, Jartti T, Ruuskanen O. Nasal swabs for detection of
respiratory syncytial virus RNA in children. Arch Dis Child 2007.
147. Whiley DM, Syrmis MW, Mackay IM, Sloots TP. Detection of Human Respiratory
Syncytial Virus in Respiratory Samples by LightCycler Reverse Transcriptase PCR. J Clin
Microbiol 2002;40(12):4418-4422.
148. Wilkesmann A, Schildgen O, Eis-Hubinger AM, Geikowski T, Glatzel T, Lentze MJ, Bode
U, Simon A. Human metapneumovirus infections cause similar symptoms and clinical
severity as respiratory syncytial virus infections. Eur J Pediatr 2006;165(7):467-475.
149. Williams JV, Harris PA, Tollefson SJ, Halburnt-Rush LL, Pingsterhaus JM, Edwards KM,
Wright PF, Crowe JE, Jr. Human metapneumovirus and lower respiratory tract disease in
otherwise healthy infants and children. N Engl J Med 2004;350(5):443-450.
150. Xatzipsalti M, Kyrana S, Tsolia M, Psarras S, Bossios A, Laza-Stanca V, Johnston SL,
Papadopoulos NG. Rhinovirus viremia in children with respiratory infections. Am J Respir
Crit Care Med 2005;172(8):1037-1040.
151. Xepapadaki P, Psarras S, Bossios A, Tsolia M, Gourgiotis D, Liapi-Adamidou G,
Constantopoulos AG, Kafetzis D, Papadopoulos NG. Human Metapneumovirus as a
causative agent of acute bronchiolitis in infants. J Clin Virol 2004;30(3):267-270.
61
11. Appendix. Health diary
Name:_______________________________ Study no._______ Next appointment:_________________
Symptoms of the child in January (Mark every date):
01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
No symptoms
General malaise
Nasal discharge/runny nose
Cough
Fever/feels hot
Hoarseness
Conjunctivitis
Rash/eczema
Fast breathing
Wheezing
Diarrhoea (>3 stools/day)
Vomiting
Reduced appetite
Medicine*
Doctor's visit#
Hospital admission#
*Type of medicine:
#Reason for doctor's visit or hospital admission: